Sequential analysis of biological samples

ABSTRACT

Methods for detecting multiple targets in a biological sample are provided. The methods includes contacting the sample with a first probe; physically binding the first probe to a first target; observing a first signal from the first probe; applying a chemical agent to modify the first signal; contacting the sample with a second probe; physically binding the second probe to a second target; and observing a second signal from the second probe. The methods disclosed herein also provide for multiple iterations of binding, observing, signal modification for deriving information about multiple targets in a single sample. An associated kit and device are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Divisional of U.S. patent application Ser. No. 11/560,599,which was filed on Nov. 16, 2006, and entitled SEQUENTIAL ANALYSIS OFBIOLOGICAL SAMPLES, which is hereby incorporated by reference in itsentirety.

BACKGROUND

Disclosed herein are methods for sequentially analyzing a biologicalsample to discern characteristics of the sample, for example, thepresence, absence, concentration, and/or spatial distribution ofmultiple biological targets in a biological sample.

Various methods may be used in biology and in medicine to observedifferent targets in a biological sample. For example, analysis ofproteins in histological sections and other cytological preparations maybe performed using the techniques of histochemistry,immunohistochemistry (IHC), or immunofluorescence.

Many of the current techniques may detect only a few targets at one time(such as, IHC where number of targets detectable is limited by theflorescence-based detection system) in a single sample. Further analysisof targets may require use of additional biological samples from thesource limiting the ability to determine relative characteristics of thetargets such as the presence, absence, concentration, and/or the spatialdistribution of multiple biological targets in the biological sample.Moreover, in certain instances, a limited amount of sample may beavailable for analysis or the individual sample may require furtheranalysis. Thus, methods, agents, and devices capable of iterativelyanalyze an individual sample are needed.

BRIEF DESCRIPTION

In some embodiments, methods of detecting multiple targets in abiological sample are provided. The methods include the steps ofcontacting the sample with a first probe, physically binding the firstprobe to a first target, observing a first signal from the first probe,applying a chemical agent to modify the first signal, contacting thesample with a second probe, physically binding the second probe to asecond target, and observing a second signal from the second probe. Theprocess of contacting, binding, observing and modifying may beiteratively repeated.

In some embodiments, kits for detection of multiple targets in abiological sample are provided. The kit includes a first probe capableof binding to a first target and providing a first signal and a secondprobe capable of binding to a second target and providing a secondsignal. The first probe is responsive to a chemical agent to result inmodification of the first signal. The kits provided herein may alsoinclude additional (e.g., second, third, or n^(th)) probes capable ofbinding to a target and providing multiple signals.

In some embodiments, devices including a sample handling system, areagent dispensing system, and a signal detection system are provided.The device may be employed to detect multiple targets in a biologicalsample using the method including the steps of: contacting the samplewith a first probe, physically binding the first probe to a firsttarget, observing a first signal from the first probe, applying achemical agent to modify the first signal, contacting the sample with asecond probe, physically binding the second probe to a second target,and observing a second signal from the second probe.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is the absorbance spectra of Sample 1 as a function ofwavelength, after 10 minutes and 15 minutes.

FIG. 2 is the absorbance spectra for Samples 1, 2, and 3 as a functionof wavelength.

FIG. 3 is the absorbance spectra of Sample 4 as a function ofwavelength, after 30 minutes and 140 minutes.

FIG. 4 is the absorbance spectra of Sample 5 as a function ofwavelength, after 20 minutes, 60 minutes, and 210 minutes.

FIG. 5 is the absorbance spectra of Sample 6 as a function ofwavelength, after 12 minutes and 16 minutes.

FIG. 6 is the absorbance spectra of Sample 8 as a function ofwavelength, after 22 minutes, 70 minutes, and 210 minutes.

FIG. 7 is the absorbance spectra of Samples 9a, 10a, and 11a as afunction of wavelength.

FIG. 8 is the absorbance spectra of Samples 9b and 10b as a function ofwavelength.

FIG. 9 is the absorbance spectra of Samples 12a and 12b as a function ofwavelength.

FIG. 10 is the absorbance spectra of Samples 13, 14, and 15 as afunction of wavelength.

FIG. 11 is the absorbance spectra of Samples 16 and 17 as a function ofwavelength.

FIG. 12 depicts micrographs (at 10× magnification) of Sample 18a (beforesignal modification) and Sample 18b (after signal modification).

FIG. 13 depicts micrographs (at 10× magnification) of Sample 19a (beforesignal modification) and Sample 19b (after signal modification).

FIG. 14 depicts micrographs of Sample 20a (before signal modification)and Sample 20b (after signal modification).

FIG. 15 depicts micrographs of Samples 21a and b (before signalmodification) and Sample 21c (after signal modification).

FIG. 16 depicts micrographs of Samples 22a and b (before signalmodification) and Sample 22c (after signal modification).

FIG. 17 depicts micrographs of Samples 23a-e.

FIG. 18 depicts micrographs of Sample 24a (before signal modification)and Sample 24b (after signal modification).

FIG. 19 depicts micrographs of Sample 25a (Cy3 and Cy5 channels), Sample25b (Cy3 and Cy5 channels), and Samples 25c-25j.

FIG. 20 depicts micrographs of Samples 26a-h.

FIG. 21 depicts the micrographs of Samples 27a-c.

FIG. 22 is a plot of average pixel intensity of the background for eachcycle in the imaging in Example 20.

FIG. 23 shows comparison between micrographs of Samples 28a-c and 29a-c.

FIG. 24 depicts micrographs of Samples 30a-d.

FIG. 25 depicts micrographs of Samples 30c-f.

FIG. 26 is a plot of average pixel intensities for the background ofeach cycle for Samples 30c and 30d.

FIG. 27 depicts micrographs of Samples 31a, 31b, and 31c.

FIG. 28 depicts micrographs of Samples 32a, 32b, and 32c.

DETAILED DESCRIPTION

To more clearly and concisely describe and point out the subject matterof the claimed invention, the following definitions are provided forspecific terms, which are used in the following description and theappended claims.

The singular forms “a” “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Approximating language, as usedherein throughout the specification and claims, may be applied to modifyany quantitative representation that could permissibly vary withoutresulting in a change in the basic function to which it is related.Accordingly, a value modified by a term such as “about” is not to belimited to the precise value specified. Unless otherwise indicated, allnumbers expressing quantities of ingredients, properties such asmolecular weight, reaction conditions, so forth used in thespecification and claims are to be understood as being modified in allinstances by the term “about.” Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the followingspecification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least each numerical parameter should atleast be construed in light of the number of reported significant digitsand by applying ordinary rounding techniques.

“Biological sample” as used herein, refers to a sample obtained from abiological subject, including sample of biological tissue or fluidorigin obtained in vivo or in vitro. Such samples can be, but are notlimited to, body fluid (e.g., blood, blood plasma, serum, or urine),organs, tissues, fractions and cells isolated from mammals including,humans. Biological samples also may include sections of the biologicalsample including tissues (e.g., sectional portions of an organ ortissue). Biological samples may also include extracts from a biologicalsample, for example, an antigen from a biological fluid (e.g., blood orurine). Biological samples may be dispersed in solution or may beimmobilized on a solid support, such as in blots, assays, arrays, glassslides, microtiter, or ELISA plates.

A biological sample may be of prokaryotic origin or eukaryotic origin(e.g., insects, protozoa, birds, fish, reptiles). In some embodiments,the biological sample is mammalian (e.g., rat, mouse, cow, dog, donkey,guinea pig, or rabbit). In certain embodiments, the biological sample isof primate origin (e.g., example, chimpanzee or human).

“Target,” as used herein, generally refers to the component of abiological sample that may be detected when present in the biologicalsample. The target may be any substance for which there exists anaturally occurring specific binder (e.g., an antibody), or for which aspecific binder may be prepared (e.g., a small molecule binder). Ingeneral, the binder portion of the probe may bind to target through oneor more discrete chemical moieties of the target or a three-dimensionalstructural component of the target (e.g., 3D structures resulting frompeptide folding). The target may include one or more of peptides,proteins (e.g., antibodies, affibodies, or aptamers), nucleic acids(e.g., polynucleotides, DNA, RNA, or aptamers); polysaccharides (e.g.,lectins or sugars), lipids, enzymes, enzyme substrates, ligands,receptors, antigens, or haptens.

As used herein, the term “antibody” refers to an immunoglobulin thatspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of another molecule. Theantibody may be monoclonal or polyclonal and may be prepared bytechniques that are well known in the art such as immunization of a hostand collection of sera (polyclonal) or by preparing continuous hybridcell lines and collecting the secreted protein (monoclonal), or bycloning and expressing nucleotide sequences or mutagenized versionsthereof coding at least for the amino acid sequences required forspecific binding of natural antibodies. Antibodies may include acomplete immunoglobulin or fragment thereof, which immunoglobulinsinclude the various classes and isotypes, such as IgA, IgD, IgE, IgG1,IgG2a, IgG2b and IgG3, IgM. Functional antibody fragments may includeportions of an antibody capable of retaining binding at similar affinityto full-length antibody (for example, Fab, Fv and F(ab′)₂, or Fab′). Inaddition, aggregates, polymers, and conjugates of immunoglobulins ortheir fragments may be used where appropriate so long as bindingaffinity for a particular molecule is substantially maintained.

As used herein, the term “probe” refers to an agent including a binderand a signal generator. In some embodiments, the binder and the signalgenerator of the probe are embodied in a single entity (e.g., aradioactive or fluorescent molecule capable of binding a target). Inalternative embodiments, the binder and the signal generator areembodied in discrete entities (e.g., a primary antibody capable ofbinding target and labeled secondary antibody capable of binding theprimary antibody).

When the binder and signal generator are separate entities they may beapplied to a biological sample in a single step or separate steps. Thus,the binder and signal generator may be attached directly (e.g., via aradiolabeled atom incorporated into the binder) or indirectly (e.g.,through a linker, which may include a cleavage site) and applied to thebiological sample in a single step. For some embodiments in which thebinder and the signal generator are separate entities, they may bepre-attached prior to application to the biological sample and appliedto the biological sample in a single step. In other embodiments in whichthe binder and signal generator are separate entities, they may beapplied to the biological sample independently and associate followingapplication.

As used herein, the term “binder” refers to a biological molecule thatmay non-covalently bind to one or more targets in the biological sample.A binder may specifically bind to a target. Suitable binders may includeone or more of natural or modified peptides, proteins (e.g., antibodies,affibodies, or aptamers), nucleic acids (e.g., polynucleotides, DNA,RNA, or aptamers); polysaccharides (e.g., lectins, sugars), lipids,enzymes, enzyme substrates or inhibitors, ligands, receptors, antigens,haptens, and the like. A suitable binder may be selected depending onthe sample to be analyzed and the targets available for detection. Forexample, a target in the sample may include a ligand and the binder mayinclude a receptor or a target may include a receptor and the probe mayinclude a ligand. Similarly, a target may include an antigen and thebinder may include an antibody or antibody fragment or vice versa. Insome embodiments, a target may include a nucleic acid and the binder mayinclude a complementary nucleic acid. In some embodiments, both thetarget and the binder may include proteins capable of binding to eachother.

As used herein, the term “signal generator” refers to a molecule capableof providing a detectable signal using one or more detection techniques(e.g., spectrometry, calorimetry, spectroscopy, or visual inspection).Suitable examples of a detectable signal may include an optical signal,and electrical signal, or a radioactive signal. Examples of signalgenerators useful in the inventive methods include, for example, achromophore, a fluorophore, a Raman-active tag, a radioactive label, anenzyme, an enzyme substrate, or combinations thereof. As stated above,with regard to the probe, the signal generator and the binder may bepresent in a single entity (e.g., a target binding protein with afluorescent label or radiolabel). And, in other embodiments the binderand the signal generator are discrete entities (e.g., target receptorprotein and antibody against the that particular receptor protein) thatassociate with each other prior to or upon introduction to the sample.

As used herein, the term “fluorophore” refers to a chemical compound,which when excited by exposure to a particular wavelength of light,emits light (at a different wavelength. Fluorophores may be described interms of their emission profile, or “color.” Green fluorophores (forexample Cy3, FITC, and Oregon Green) may be characterized by theiremission at wavelengths generally in the range of 515-540 nanometers.Red fluorophores (for example Texas Red, Cy5, and tetramethylrhodamine)may be characterized by their emission at wavelengths generally in therange of 590-690 nanometers. Examples of fluorophores include, but arenot limited to, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonicacid, acridine, derivatives of acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-(3-vinylsulfonyl)phenyl)naphthalimide-3,5 disulfonate (Lucifer YellowVS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BrilliantYellow, coumarin, coumarin derivatives, 7-amino-4-methylcoumarin (AMC,Coumarin 120), 7-amino-trifluoromethylcouluarin (Coumaran 151),cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI),5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red),7-diethylamino-3-(4′-isothiocyanatophenyl)4-methylcoumarin, -,4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid,4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid,5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride),eosin, derivatives of eosin such as eosin isothiocyanate, erythrosine,derivatives of erythrosine such as erythrosine B and erythrosinisothiocyanate; ethidium; fluorescein and derivatives such as5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein(DTAF), 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),fluorescein, fluorescein isothiocyanate (FITC), QFITC (XRITC);fluorescamine derivative (fluorescent upon reaction with amines); IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red,B-phycoerythrin; o-phthaldialdehyde derivative (fluorescent uponreaction with amines); pyrene and derivatives such as pyrene, pyrenebutyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron.RTM. Brilliant Red 3B-A), rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl Rhodamine,tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acidand lathanide chelate derivatives, quantum dots, cyanines, andsquaraines.

As used herein, the term “specific binding” refers to the specificrecognition of one of two different molecules for the other compared tosubstantially less recognition of other molecules. The molecules mayhave areas on their surfaces or in cavities giving rise to specificrecognition between the two molecules arising from one or more ofelectrostatic interactions, hydrogen bonding, or hydrophobicinteractions. Specific binding examples include, but are not limited to,antibody-antigen interactions, enzyme-substrate interactions,polynucleotide interactions, and the like. In some embodiments, a bindermolecule may have an intrinsic equilibrium association constant (KA) forthe target no lower than about 10⁵ M⁻¹ under ambient conditions (i.e., apH of about 6 to about 8 and temperature ranging from about 0° C. toabout 37° C.).

As used herein, the term “in situ” generally refers to an eventoccurring in the original location, for example, in intact organ ortissue or in a representative segment of an organ or tissue. In someembodiments, in situ analysis of targets may be performed on cellsderived from a variety of sources, including an organism, an organ,tissue sample, or a cell culture. In situ analysis provides contextualinformation that may be lost when the target is removed from its site oforigin. Accordingly, in situ analysis of targets describes analysis oftarget-bound probe located within a whole cell or a tissue sample,whether the cell membrane is fully intact or partially intact wheretarget-bound probe remains within the cell. Furthermore, the methodsdisclosed herein may be employed to analyze targets in situ in cell ortissue samples that are fixed or unfixed.

The invention includes embodiments that relate generally to methodsapplicable in analytical, diagnostic, or prognostic applications such asanalyte detection, histochemistry, immunohistochemistry, orimmunofluorescence. In some embodiments, the methods disclosed hereinmay be particularly applicable in histochemistry, immunohistochemistry,or immunofluorescence.

The disclosed methods relate generally to detection of multiple targetsin a single biological sample. In some embodiments, methods of detectingmultiple targets in a single biological sample using the same detectionchannel are disclosed. The targets may be present on the surface ofcells in suspension, on the surface of cytology smears, on the surfaceof histological sections, on the surface of DNA microarrays, on thesurface of protein microarrays, or on the surface of solid supports(such as gels, blots, glass slides, beads, or ELISA plates).

The methods disclosed herein may allow detection of a plurality oftargets in the same biological sample with little or no effect on theintegrity of the biological sample. Detecting the targets in the samebiological sample may further provide spatial information about thetargets in the biological sample. Methods disclosed herein may also beapplicable in analytical applications where a limited amount ofbiological sample may be available for analysis and the same sample mayhave to e processed for multiple analyses. Furthermore, the samedetection channel may be employed for detection of different targets inthe sample, enabling fewer chemistry requirements for analyses ofmultiple targets. The methods may further facilitate analyses based ondetection methods that may be limited in the number of simultaneouslydetectable targets because of limitations of resolvable signals. Forexample, using fluorescent-based detection, the number of targets thatmay be simultaneously detected may be limited to about four as onlyabout four fluorescent signals may be resolvable based on theirexcitation and emission wavelength properties. In some embodiments, themethods disclosed herein may allow detection of greater than fourtargets using fluorescent-based detection system.

In some embodiments, the method of detecting multiple targets in abiological sample includes sequential detection of targets in thebiological sample. The method generally includes the steps of detectinga first target in the biological sample, modifying the signal from thefirst target using a chemical agent, and detecting a second target inthe biological sample. The method may further include repeating the stepof modification of signal from the second target followed by detecting athird target in the biological sample, and so forth.

In some embodiments, the method includes the steps of contacting abiological sample with a first probe and physically binding a firstprobe to a first target. The method further includes observing a firstsignal from the first probe. A chemical agent is applied to the probe tomodify the first signal. The method further includes contacting thebiological sample with a second probe and physically binding the secondprobe to a second target in the biological sample followed by observinga second signal from the second probe.

Biological Samples

A biological sample in accordance with one embodiment of the inventionmay be solid or fluid. Suitable examples of biological samples mayinclude, but are not limited to, but are not limited to, cultures,blood, plasma, serum, saliva, cerebral spinal fluid, pleural fluid,milk, lymph, sputum, semen, urine, stool, tears, saliva, needleaspirates, external sections of the skin, respiratory, intestinal, andgenitourinary tracts, tumors, organs, cell cultures or cell cultureconstituents, or solid tissue sections. In some embodiments, thebiological sample may be analyzed as is, that is, without harvest and/orisolation of the target of interest. In an alternate embodiment, harvestand isolation of targets may be performed prior to analysis. In someembodiments, the methods disclosed herein may be particularly suitablefor in-vitro analysis of biological samples.

A biological sample may include any of the aforementioned samplesregardless of their physical condition, such as, but not limited to,being frozen or stained or otherwise treated. In some embodiments, abiological sample may include compounds which are not naturallyintermixed with the sample in nature such as preservatives,anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

In some embodiments, a biological sample may include a tissue sample, awhole cell, a cell constituent, a cytospin, or a cell smear. In someembodiments, a biological sample essentially includes a tissue sample. Atissue sample may include a collection of similar cells obtained from atissue of a biological subject that may have a similar function. In someembodiments, a tissue sample may include a collection of similar cellsobtained from a tissue of a human. Suitable examples of human tissuesinclude, but are not limited to, (1) epithelium; (2) the connectivetissues, including blood vessels, bone and cartilage; (3) muscle tissue;and (4) nerve tissue. The source of the tissue sample may be solidtissue obtained from a fresh, frozen and/or preserved organ or tissuesample or biopsy or aspirate; blood or any blood constituents; bodilyfluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid,or interstitial fluid; or cells from any time in gestation ordevelopment of the subject. In some embodiments, the tissue sample mayinclude primary or cultured cells or cell lines.

In some embodiments, a biological sample includes tissue sections ofcolon, normal breast tissue, prostate cancer, colon adenocarcinoma,breast tissue microarray, breast TMA, or normal prostrate. A tissuesection may include a single part or piece of a tissue sample, forexample, a thin slice of tissue or cells cut from a tissue sample. Insome embodiments, multiple sections of tissue samples may be taken andsubjected to analysis, provided the methods disclosed herein may be usedfor analysis of the same section of the tissue sample with respect to atleast two different targets (at morphological or molecular level). Insome embodiments, the same section of tissue sample may be analyzed withrespect to at least four different targets (at morphological ormolecular level). In some embodiments, the same section of tissue samplemay be analyzed with respect to greater than four different targets (atmorphological or molecular level). In some embodiments, the same sectionof tissue sample may be analyzed at both morphological and molecularlevels. A tissue section, if employed as a biological sample may have athickness in a range that is less than about 100 micrometers, in a rangethat is less than about 50 micrometers, in a range that is less thanabout 25 micrometers, or in range that is less than about 10micrometers.

Targets

A target according to an embodiment of the invention may be present onthe surface of a biological sample (for example, an antigen on a surfaceof a tissue section) or present in the bulk of the sample (for example,an antibody in a buffer solution). In some embodiments, a target may notbe inherently present on the surface of a biological sample and thebiological sample may have to be processed to make the target availableon the surface. In some embodiments, the target may be soluble in a bodyfluid such as blood, blood plasma, serum, or urine. In some embodiments,the target may be in a tissue, either on a cell surface, or within acell.

Suitability of target(s) to be analyzed may be determined by the typeand nature of analysis required for the biological sample. In someembodiments, a target may provide information about the presence orabsence of an analyte in the biological sample. In another embodiment, atarget may provide information on a state of a biological sample. Forexample, if the biological sample includes a tissue sample, the methodsdisclosed herein may be used to detect target(s) that may help incomparing different types of cells or tissues, comparing differentdevelopmental stages, detecting the presence of a disease orabnormality, or determining the type of disease or abnormality.

Suitable targets may include one or more of peptides, proteins (e.g.,antibodies, affibodies, or aptamers), nucleic acids (e.g.,polynucleotides, DNA, RNA, or aptamers); polysaccharides (e.g., lectinsor sugars), lipids, enzymes, enzyme substrates, ligands, receptors,antigens, or haptens. One or more of the aforementioned targets may becharacteristic of particular cells, while other targets may beassociated with a particular disease or condition. In some embodiments,targets in a tissue sample that may be detected and analyzed using themethods disclosed herein may include, but are not limited to, but arenot limited to, prognostic targets, hormone or hormone receptor targets,lymphoid targets, tumor targets, cell cycle associated targets, neuraltissue and tumor targets, or cluster differentiation targets

Suitable examples of prognostic targets may include enzymatic targetssuch as galactosyl transferase II, neuron specific enolase, protonATPase-2, or acid phosphatase.

Suitable examples of hormone or hormone receptor targets may includehuman chorionic gonadotropin (HCG), adrenocorticotropic hormone,carcinoembryonic antigen (CEA), prostate-specific antigen (PSA),estrogen receptor, progesterone receptor, androgen receptor, gC1q-R/p33complement receptor, IL-2 receptor, p75 neurotrophin receptor, PTHreceptor, thyroid hormone receptor, or insulin receptor.

Suitable examples of lymphoid targets may includealpha-1-antichymotrypsin, alpha-1-antitrypsin, B cell target, bcl-2,bcl-6, B lymphocyte antigen 36 kD, BM1 (myeloid target), BM2 (myeloidtarget), galectin-3, granzyme B, HLA class I Antigen, HLA class II (DP)antigen, HLA class II (DQ) antigen, HLA class II (DR) antigen, humanneutrophil defensins, immunoglobulin A, immunoglobulin D, immunoglobulinG, immunoglobulin M, kappa light chain, kappa light chain, lambda lightchain, lymphocyte/histocyte antigen, macrophage target, muramidase(lysozyme), p80 anaplastic lymphoma kinase, plasma cell target,secretory leukocyte protease inhibitor, T cell antigen receptor (JOVI1), T cell antigen receptor (JOVI 3), terminal deoxynucleotidyltransferase, or unclustered B cell target.

Suitable examples of tumour targets may include alpha fetoprotein,apolipoprotein D, BAG-1 (RAP46 protein), CA19-9 (sialyl lewisa), CA50(carcinoma associated mucin antigen), CA125 (ovarian cancer antigen),CA242 (tumour associated mucin antigen), chromogranin A, clusterin(apolipoprotein J), epithelial membrane antigen, epithelial-relatedantigen, epithelial specific antigen, gross cystic disease fluidprotein-15, hepatocyte specific antigen, heregulin, human gastric mucin,human milk fat globule, MAGE-1, matrix metalloproteinases, melan A,melanoma target (HMB45), mesothelin, metallothionein, microphthalmiatranscription factor (MITF), Muc-1 core glycoprotein. Muc-1glycoprotein, Muc-2 glycoprotein, Muc-5AC glycoprotein, Muc-6glycoprotein, myeloperoxidase, Myf-3 (Rhabdomyosarcoma target), Myf-4(Rhabdomyosarcoma target), MyoD1 (Rhabdomyosarcoma target), myoglobin,nm23 protein, placental alkaline phosphatase, prealbumin, prostatespecific antigen, prostatic acid phosphatase, prostatic inhibin peptide,PTEN, renal cell carcinoma target, small intestinal mucinous antigen,tetranectin, thyroid transcription factor-1, tissue inhibitor of matrixmetalloproteinase 1, tissue inhibitor of matrix metalloproteinase 2,tyrosinase, tyrosinase-related protein-1, villin, or von Willebrandfactor.

Suitable examples of cell cycle associated targets may include apoptosisprotease activating factor-1, bcl-w, bcl-x, bromodeoxyuridine, CAK(cdk-activating kinase), cellular apoptosis susceptibility protein(CAS), caspase 2, caspase 8, CPP32 (caspase-3), CPP32 (caspase-3),cyclin dependent kinases, cyclin A, cyclin B1, cyclin D1, cyclin D2,cyclin D3, cyclin E, cyclin G, DNA fragmentation factor (N-terminus),Fas (CD95), Fas-associated death domain protein, Fas ligand, Fen-1,IPO-38, Mcl-1, minichromosome maintenance proteins, mismatch repairprotein (MSH2), poly (ADP-Ribose) polymerase, proliferating cell nuclearantigen, p16 protein, p27 protein, p34cdc2, p57 protein (Kip2), p105protein, Stat 1 alpha, topoisomerase I, topoisomerase II alpha,topoisomerase III alpha, or topoisomerase II beta.

Suitable examples of neural tissue and tumor targets may include alpha Bcrystallin, alpha-internexin, alpha synuclein, amyloid precursorprotein, beta amyloid, calbindin, choline acetyltransferase, excitatoryamino acid transporter 1, GAP43, glial fibrillary acidic protein,glutamate receptor 2, myelin basic protein, nerve growth factor receptor(gp75), neuroblastoma target, neurofilament 68 kD, neurofilament 160 kD,neurofilament 200 kD, neuron specific enolase, nicotinic acetylcholinereceptor alpha4, nicotinic acetylcholine receptor beta2, peripherin,protein gene product 9, S-100 protein, serotonin, SNAP-25, synapsin I,synaptophysin, tau, tryptophan hydroxylase, tyrosine hydroxylase, orubiquitin.

Suitable examples of cluster differentiation targets may include CD1a,CD1b, CD1c, CD1d, CD1e, CD2, CD3delta, CD3epsilon, CD3gamma, CD4, CD5,CD6, CD7, CD8alpha, CD8beta, CD9, CD10, CD11a, CD11b, CD11c, CDw12,CD13, CD14, CD15, CD15s, CD16a, CD16b, CDw17, CD18, CD19, CD20, CD21,CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33,CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c,CD42d, CD43, CD44, CD44R, CD45, CD46, CD47, CD48, CD49a, CD49b, CD49c,CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57,CD58, CD59, CDw60, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s,CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72,CD73, CD74, CDw75, CDw76, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83,CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CDw93, CD94,CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105,CD106, CD107a, CD107b, CDw108, CD109, CD114, CD115, CD116, CD117,CDw119, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CDw125,CD126, CD127, CDw128a, CDw128b, CD130, CDw131, CD132, CD134, CD135,CDw136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143,CD144, CDw145, CD146, CD147, CD148, CDw149, CDw150, CD151, CD152, CD153,CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162, CD163, CD164,CD165, CD166, and TCR-zeta.

Other suitable prognostic targets hormone or hormone receptor targetslymphoid targets tumor targets cell cycle associated targets neuraltissue and tumor targets include centromere protein-F (CENP-F), giantin,involucrin, lamin A&C (XB 10), LAP-70, mucin, nuclear pore complexproteins, p180 lamellar body protein, ran, cathepsin D, Ps2 protein,Her2-neu, P53, S100, epithelial target antigen (EMA), TdT, MB2, MB3,PCNA, or Ki67.

Probes

In some embodiments, the present methods may employ probes that do notinclude an intrinsic signal generator. In some alternative embodiments,the probe does include a binder capable of binding to the target and asignal generator capable of providing a detectable signal. Thus, In someembodiments, the binder and the signal generator are not be associatedto each other and may be present as a mixture or as separate componentsthat associate following sequential application of the binder and signalgenerator to the biological sample. In alternate embodiments, the binderand the signal generator may be associated to each other. As usedherein, “associated” generally refers to two entities (for example,binder and signal generator) stably bound to one another by anyphysicochemical means. The nature of the association may be such that itdoes not substantially impair the effectiveness of either entity. Abinder and a signal generator may be associated to each other throughcovalent or non-covalent interactions. Non-covalent interactions mayinclude, but are not limited to, hydrophobic interactions, ionicinteractions, hydrogen-bond interactions, high affinity interactions(such as, biotin-avidin or biotin-streptavidin complexation), or otheraffinity interactions.

In some embodiments, a binder and a signal generator may be associatedto each other directly (that is without any linkers). In otherembodiments, a binder and a signal generator may be conjugated to eachother via a linker. A linker may include a form of linking structure orsequence formed due to the non-covalent or covalent bond formation. Insome embodiments, the linker may be chemically stable, that is, maymaintain its integrity in the presence of a chemical agent. In someembodiments, the linker may be susceptible to chemical agents that ismay be capable of dissociating, cleaving, or hydrolyzing in the presenceof a chemical agent. Suitable examples of linkers may include disulfidebonds (e.g., SPDP or SMPT), pH sensitive structures/sequences,structures/sequences that may be reduced in the presence of an reducingagent, structures/sequences that may be oxidized in the presence of anoxidizing agent, or any other chemical or physical bond that may beeasily manipulated (dissociated, cleaved, or hydrolyzed) in the presenceof a chemical agent.

In some embodiments, the binder may be intrinsically labeled with asignal generator (for example, if the binder is a protein, duringsynthesis using a detectably labeled amino acid). A binder that isintrinsically labeled may not require a separate signal generator inorder to be detected. Rather the intrinsic label may be sufficient forrendering the probe detectable. In alternate embodiments, the binder maybe labeled by binding to it a specific signal generator (i.e.,extrinsically labeled).

A binder and a signal generator may be chemically linked to each otherthrough functional groups capable of reacting and forming a linkageunder suitable conditions. Suitable examples of functional groupcombinations may include, but are not limited to, amine ester and aminesor anilines; acyl azide and amines or anilines; acyl halides and amines,anilines, alcohols, or phenols; acyl nitrile and alcohols or phenols;aldehyde and amines or anilines; alkyl halide and amines, anilines,alcohols, phenols or thiols; alkyl sulfonate and thiols, alcohols orphenols; anhydride and alcohols, phenols, amines or anilines; arylhalide and thiols; aziridine and thiols or thioethers; carboxylic acidand amines, anilines, alcohols or alkyl halides; diazoalkane andcarboxylic acids; epoxide and thiols; haloacetamide and thiols;halotriazin and amines, anilines or phenols; hydrazine and aldehydes orketones; hydroxyamine and aldehydes or ketones; imido ester and aminesor anilines; isocyanate and amines or anilines; and isothiocyanate andamines or anilines. A functional group in one of the aforementionedfunctional group pair may be present in a binder and a correspondingfunctional group may be present in the signal generator. For example, abinder may include a carboxylic acid and the signal generator mayinclude an amine, aniline, alcohol or acyl halide, or vice versa.Conjugation between the binder and the signal generator may be effectedin this case by formation of an amide or an ester linkage.

Binders

The methods disclosed herein involve the use of binders that physicallybind to the target in a specific manner. In some embodiments, a bindermay bind to a target with sufficient specificity, that is, a binder maybind to a target with greater affinity than it does to any othermolecule. In some embodiments, the binder may bind to other molecules,but the binding may be such that the non-specific binding may be at ornear background levels. In some embodiments, the affinity of the binderfor the target of interest may be in a range that is at least 2-fold, atleast 5-fold, at least 10-fold, or more than its affinity for othermolecules. In some embodiments, binders with the greatest differentialaffinity may be employed, although they may not be those with thegreatest affinity for the target.

Binding between the target and the binder may be affected by physicalbinding. Physical binding may include binding effected usingnon-covalent interactions. Non-covalent interactions may include, butare not limited to, hydrophobic interactions, ionic interactions,hydrogen-bond interactions, or affinity interactions (such as,biotin-avidin or biotin-streptavidin complexation). In some embodiments,the target and the binder may have areas on their surfaces or incavities giving rise to specific recognition between the two resultingin physical binding. In some embodiments, a binder may bind to abiological target based on the reciprocal fit of a portion of theirmolecular shapes.

Binders and their corresponding targets may be considered as bindingpairs, of which non-limiting examples include immune-type binding-pairs,such as, antigen/antibody, antigen/antibody fragment, orhapten/anti-hapten; nonimmune-type binding-pairs, such as biotin/avidin,biotin/streptavidin, folic acid/folate binding protein, hormone/hormonereceptor, lectin/specific carbohydrate, enzyme/enzyme, enzyme/substrate,enzyme/substrate analog, enzyme/pseudo-substrate (substrate analogs thatcannot be catalyzed by the enzymatic activity), enzyme/co-factor,enzyme/modulator, enzyme/inhibitor, or vitamin B12/intrinsic factor.Other suitable examples of binding pairs may include complementarynucleic acid fragments (including DNA sequences, RNA sequences, PNAsequences, and peptide nucleic acid sequences); Protein A/antibody;Protein G/antibody; nucleic acid/nucleic acid binding protein; orpolynucleotide/polynucleotide binding protein.

In some embodiments, the binder may be a sequence- or structure-specificbinder, wherein the sequence or structure of a target recognized andbound by the binder may be sufficiently unique to that target.

In some embodiments, the binder may be structure-specific and mayrecognize a primary, secondary, or tertiary structure of a target. Aprimary structure of a target may include specification of its atomiccomposition and the chemical bonds connecting those atoms (includingstereochemistry), for example, the type and nature of linear arrangementof amino acids in a protein. A secondary structure of a target may referto the general three-dimensional form of segments of biomolecules, forexample, for a protein a secondary structure may refer to the folding ofthe peptide “backbone” chain into various conformations that may resultin distant amino acids being brought into proximity with each other.Suitable examples of secondary structures may include, but are notlimited to, alpha helices, beta pleated sheets, or random coils. Atertiary structure of a target may be is its overall three dimensionalstructure. A quaternary structure of a target may be the structureformed by its noncovalent interaction with one or more other targets ormacromolecules (such as protein interactions). An example of aquaternary structure may be the structure formed by the four-globinprotein subunits to make hemoglobin. A binder in accordance with theembodiments of the invention may be specific for any of theafore-mentioned structures.

An example of a structure-specific binder may include a protein-specificmolecule that may bind to a protein target. Examples of suitableprotein-specific molecules may include antibodies and antibodyfragments, nucleic acids (for example, aptamers that recognize proteintargets), or protein substrates (non-catalyzable).

In some embodiments, a target may include an antigen and a binder mayinclude an antibody. A suitable antibody may include monoclonalantibodies, polyclonal antibodies, multispecific antibodies (forexample, bispecific antibodies), or antibody fragments so long as theybind specifically to a target antigen.

In some embodiments, a target may include a monoclonal antibody. A“monoclonal antibody” may refer to an antibody obtained from apopulation of substantially homogeneous antibodies, that is, theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies may be highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to(polyclonal) antibody preparations that typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody may be directed against a single determinant on theantigen. A monoclonal antibody may be prepared by any known method suchas the hybridoma method, by recombinant DNA methods, or may be isolatedfrom phage antibody libraries.

In some embodiments, a biological sample may include a cell or a tissuesample and the methods disclosed herein may be employed inimmunohistochemistry (IHC). Immunochemistry may involve binding of atarget antigen to an antibody-based binder to provide information aboutthe tissues or cells (for example, diseased versus normal cells).Examples of antibodies (and the corresponding diseases/disease cells)suitable as binders for methods disclosed herein include, but are notlimited to, anti-estrogen receptor antibody (breast cancer),anti-progesterone receptor antibody (breast cancer), anti-p53 antibody(multiple cancers), anti-Her-2/neu antibody (multiple cancers),anti-EGFR antibody (epidermal growth factor, multiple cancers),anti-cathepsin D antibody (breast and other cancers), anti-Bcl-2antibody (apoptotic cells), anti-E-cadherin antibody, anti-CA125antibody (ovarian and other cancers), anti-CA15-3 antibody (breastcancer), anti-CA19-9 antibody (colon cancer), anti-c-erbB-2 antibody,anti-P-glycoprotein antibody (MDR, multi-drug resistance), anti-CEAantibody (carcinoembryonic antigen), anti-retinoblastoma protein (Rb)antibody, anti-ras oneoprotein (p21) antibody, anti-Lewis X (also calledCD15) antibody, anti-Ki-67 antibody (cellular proliferation), anti-PCNA(multiple cancers) antibody, anti-CD 3 antibody (T-cells), anti-CD4antibody (helper T cells), anti-CD5 antibody (T cells), anti-CD7antibody (thymocytes, immature T cells, NK killer cells), anti-CD8antibody (suppressor T cells), anti-CD9/p24 antibody (ALL), anti-CD10(also called CALLA) antibody (common acute lymphoblasic leukemia),anti-CD11c antibody (Monocytes, granulocytes, AML), anti-CD13 antibody(myelomonocytic cells, AML), anti-CD14 antibody (mature monocytes,granulocytes), anti-CD15 antibody (Hodgkin's disease), anti-CD19antibody (B cells), anti-CD20 antibody (B cells), anti-CD 22 antibody (Bcells), anti-CD23 antibody (activated B cells, CLL), anti-CD30 antibody(activated T and B cells, Hodgkin's disease), anti-CD31 antibody(angiogenesis marker), anti-CD33 antibody (myeloid cells, AML),anti-CD34 antibody (endothelial stem cells, stromal tumors), anti-CD35antibody (dendritic cells), anti-CD38 antibody (plasma cells, activatedT, B, and myeloid cells), anti-CD 41 antibody (platelets,megakaryocytes), anti-LCA/CD45 antibody (leukocyte common antigen),anti-CD45RO antibody (helper, inducer T cells), anti-CD45RA antibody (Bcells), anti-CD39, CD100 antibody, anti-CD95/Fas antibody (apoptosis),anti-CD99 antibody (Ewings Sarcoma marker, MIC2 gene product),anti-CD106 antibody (VCAM-1; activated endothelial cells),anti-ubiquitin antibody (Alzheimer's disease), anti-CD71 (transferrinreceptor) antibody, anti-c-myc (oncoprotein and a hapten) antibody,anti-cytokeratins (transferrin receptor) antibody, anti-vimentins(endothelial cells) antibody (B and T cells), anti-HPV proteins (humanpapillomavirus) antibody, anti-kappa light chains antibody (B cell),anti-lambda light chains antibody (B cell), anti-melanosomes (HMB45)antibody (melanoma), anti-prostate specific antigen (PSA) antibody(prostate cancer), anti-S-100 antibody (melanoma, salvary, glial cells),anti-tau antigen antibody (amyloid associated disease), anti-fibrinantibody (epithelial cells), anti-keratins antibody, anti-cytokeratinantibody (tumor), anti-alpha-catenin (cell membrane), or anti-Tn-antigenantibody (colon carcinoma, adenocarcinomas, and pancreatic cancer).

Other specific examples of suitable antibodies may include, but are notlimited to, but are not limited to, anti proliferating cell nuclearantigen, clone pc10 (Sigma Aldrich, P8825); anti smooth muscle alphaactin (SmA), clone 1A4 (Sigma, A2547); rabbit anti beta catenin (Sigma,C 2206); mouse anti pan cytokeratin, clone PCK-26 (Sigma, C1801); mouseanti estrogen receptor alpha, clone 1D5 (DAKO, M 7047); beta cateninantibody, clone 15B8 (Sigma, C 7738); goat anti vimentin (Sigma, V4630);cycle androgen receptor clone AR441 (DAKO, M3562); Von Willebrand Factor7, keratin 5, keratin 8/18, e-cadherin, Her2/neu, Estrogen receptor,p53, progesterone receptor, beta catenin; donkey anti-mouse (JacksonImmunoresearch, 715-166-150); or donkey anti rabbit (JacksonImmunoresearch, 711-166-152).

In some embodiments, a binder may be sequence-specific. Asequence-specific binder may include a nucleic acid and the binder maybe capable of recognizing a particular linear arrangement of nucleotidesor derivatives thereof in the target. In some embodiments, the lineararrangement may include contiguous nucleotides or derivatives thereofthat may each bind to a corresponding complementary nucleotide in thebinder. In an alternate embodiment, the sequence may not be contiguousas there may be one, two, or more nucleotides that may not havecorresponding complementary residues on the probe. Suitable examples ofnucleic acid-based binders may include, but are not limited to, DNA orRNA oligonucleotides or polynucleotides. In some embodiments, suitablenucleic acids may include nucleic acid analogs, such as dioxygenin dCTP,biotin dcTP 7-azaguanosine, azidothymidine, inosine, or uridine.

In certain embodiments, both the binder and the target may includenucleic acids. In some embodiments, a nucleic-acid based binder may forma Watson-Crick bond with the nucleic acid target. In another embodiment,the nucleic acid binder may form a Hoogsteen bond with the nucleic acidtarget, thereby forming a triplex. A nucleic acid binder that binds byHoogsteen binding may enter the major groove of a nucleic acid targetand hybridizes with the bases located there. Suitable examples of theabove binders may include molecules that recognize and bind to the minorand major grooves of nucleic acids (for example, some forms ofantibiotics.) In certain embodiments, the nucleic acid binders may formboth Watson-Crick and Hoogsteen bonds with the nucleic acid target (forexample, bis PNA probes are capable of both Watson-Crick and Hoogsteenbinding to a nucleic acid).

The length of nucleic acid binder may also determine the specificity ofbinding. The energetic cost of a single mismatch between the binder andthe nucleic acid target may be relatively higher for shorter sequencesthan for longer ones. In some embodiments, hybridization of smallernucleic acid binders may be more specific than the hybridization oflonger nucleic acid probes, as the longer probes may be more amenable tomismatches and may continue to bind to the nucleic acid depending on theconditions. In certain embodiments, shorter binders may exhibit lowerbinding stability at a given temperature and salt concentration. Bindersthat may exhibit greater stability to bind short sequences may beemployed in this case (for examples, bis PNA). In some embodiments, thenucleic acid binder may have a length in range of from about 4nucleotides to about 12 nucleotides, from about 12 nucleotides to about25 nucleotides, from about 25 nucleotides to about 50 nucleotides, fromabout 50 nucleotides to about 100 nucleotides, from about 100nucleotides to about 250 nucleotides, from about 250 nucleotides toabout 500 nucleotides, or from about 500 nucleotides to about 1000nucleotides. In some embodiments, the nucleic acid binder may have alength in a range that is greater than about 1000 nucleotides.Notwithstanding the length of the nucleic acid binder, all thenucleotide residues of the binder may not hybridize to complementarynucleotides in the nucleic acid target. For example, the binder mayinclude 50 nucleotide residues in length, and only 25 of thosenucleotide residues may hybridize to the nucleic acid target. In someembodiments, the nucleotide residues that may hybridize may becontiguous with each other. The nucleic acid binders may be singlestranded or may include a secondary structure. In some embodiments, abiological sample may include a cell or a tissue sample and thebiological sample may be subjected to in-situ hybridization (ISH) usinga nucleic acid binder. In some embodiments, a tissue sample may besubjected to in-situ hybridization in addition to immunohistochemistry(IHC) to obtain desired information regarding the tissue sample.

Regardless of the type of binder and the target, the specificity ofbinding between the binder and the target may also be affected dependingon the binding conditions (for example, hybridization conditions in caseof complementary nucleic acids. Suitable binding conditions may berealized by modulation one or more of pH, temperature, or saltconcentration.

As noted hereinabove, a binder may be intrinsically labeled (signalgenerator attached during synthesis of binder) with a signal generatoror extrinsically labeled (signal generator attached during a laterstep). For example for a protein-based binder, an intrinsically labeledbinder may be prepared by employing labeled amino acids. Similarly, anintrinsically labeled nucleic acid may be synthesized using methods thatincorporate signal generator-labeled nucleotides directly into thegrowing nucleic acid. In some embodiments, a binder may be synthesizedin a manner such that signal generators may e incorporated at a laterstage. For example, this latter labeling may be accomplished by chemicalmeans by the introduction of active amino or thiol groups into nucleicacids of peptide chains. In some embodiments, a binder such a protein(for example, an antibody) or a nucleic acid (for example, a DNA) may bedirectly chemically labeled using appropriate chemistries for the same.

In some embodiments, combinations of binders may be used that mayprovide greater specificity or in certain embodiments amplification ofthe signal. Thus, In some embodiments, a sandwich of binders may beused, where the first binder may bind to the target and serve to providefor secondary binding, where the secondary binder may or may not includea signal generator, which may further provide for tertiary binding (ifrequired) where the tertiary binding member may include a signalgenerator.

Suitable examples of binder combinations may include primaryantibody-secondary antibody, complementary nucleic acids, or otherligand-receptor pairs (such as biotin-streptavidin). Some specificexamples of suitable binder pairs may include mouse anti-myc forrecombinant expressed proteins with c-myc epitope; mouse anti-H HisG forrecombinant protein with His-Tag epitope, mouse anti-xpress forrecombinant protein with epitope-tag, rabbit anti-goat for goat IgGprimary molecules, complementary nucleic acid sequence for a nucleicacid; mouse anti-thio for thioredoxin fusion proteins, rabbit anti-GFPfor fusion protein, jacalin for α-D-galactose; and melibiose forcarbohydrate-binding proteins, sugars, nickel couple matrix or heparin.

In some embodiments, a combination of a primary antibody and a secondaryantibody may be used as a binder. A primary antibody may be capable ofbinding to a specific region of the target and the secondary antibodymay be capable of binding to the primary antibody. A secondary antibodymay be attached to a signal generator before binding to the primaryantibody or may be capable of binding to a signal generator at a laterstep. In an alternate embodiment, a primary antibody and specificbinding ligand-receptor pairs (such as biotin-streptavidin) may be used.The primary antibody may be attached to one member of the pair (forexample biotin) and the other member (for example streptavidin) may belabeled with a signal generator. The secondary antibody, avidin,streptavidin, or biotin may be each independently labeled with a signalgenerator.

In some embodiments, the methods disclosed herein may be employed in animmunohistochemical procedure, and a primary antibody may be used tospecifically bind the target antigen in the tissue sample. A secondaryantibody may be used to specifically bind to the primary antibody,thereby forming a bridge between the primary antibody and a subsequentreagent (for example a signal generator), if any. For example, a primaryantibody may be mouse IgG (an antibody created in mouse) and thecorresponding secondary antibody may be goat anti-mouse (antibodycreated in goat) having regions capable of binding to a region in mouseIgG.

In some embodiments, signal amplification may be obtained when severalsecondary antibodies may bind to epitopes on the primary antibody. In animmunohistochemical procedure a primary antibody may be the firstantibody used in the procedure and the secondary antibody may be thesecond antibody used in the procedure. In some embodiments, a primaryantibody may be the only antibody used in an IHC procedure.

Signal Generators

The type of signal generator suitable for the methods disclosed hereinmay depend on a variety of factors, including the nature of the analysisbeing conducted, the type of the energy source and detector used, thetype of chemical agent employed, the type of binder, the type of target,or the mode of attachment between the binder and the signal generator(e.g., cleavable or non-cleavable).

A suitable signal generator may include a molecule or a compound capableof providing a detectable signal. A signal generator may provide acharacteristic signal following interaction with an energy source or acurrent. An energy source may include electromagnetic radiation sourceand a fluorescence excitation source. Electromagnetic radiation sourcemay be capable of providing electromagnetic energy of any wavelengthincluding visible, infrared, and ultraviolet. Electromagnetic radiationmay be in the form of a direct light source or may be emitted by a lightemissive compound such as a donor fluorophore. A fluorescence excitationsource may be capable of making a source fluoresce or may give rise tophotonic emissions (that is, electromagnetic radiation, directedelectric field, temperature, physical contact, or mechanicaldisruption). Suitable signal generators may provide a signal capable ofbeing detected by a variety of methods including optical measurements(for example, fluorescence), electrical conductivity, or radioactivity.Suitable signal generators may be, for example, light emitting, energyaccepting, fluorescing, radioactive, or quenching.

A suitable signal generator may be sterically and chemically compatiblewith the constituents to which it is bound, for example, a binder.Additionally, a suitable signal generator may not interfere with thebinding of the binder to the target, nor may it affect the bindingspecificity of the binder. A suitable signal generator may be organic orinorganic in nature. In some embodiments, a signal generator may be of achemical, peptide or nucleic acid nature.

A signal generator may be directly or indirectly detectable. A directlydetectable moiety may be one that may be detected directly by itsability to emit a signal, such as for example a fluorescent label thatemits light of a particular wavelength following excitation by light ofanother lower, characteristic wavelength and/or absorb light of aparticular wavelength. An indirectly detectable signal generator may beone that may be detected indirectly by its ability to bind, recruit,and, in some cases, cleave another moiety, which may in turn emit asignal. An example of an indirectly detectable signal generator may bean enzyme-based signal generator, which when contacted with a suitablesubstrate may cleave the substrate to provide a detectable signal.Alternatively, an indirectly detectable signal generator may be capableof binding a compound that does emit a signal. For example, a signalgenerator, such as, biotin which itself does not emit a signal whenbound to labeled avidin or streptavidin molecules may be detected. Otherexamples of indirectly detectable signal generators may include ligandsthat bind specifically to particular receptors. Detectably labeledreceptors may be allowed to bind to ligand labeled binders in order todetect the binders. For example, an antibody-based binder may beattached a small hapten and a signal generator may be attached to ananti-hapten antibody that may bind specifically to hapten.

A signal-generator, suitable in accordance with the methods disclosedherein may be amenable to manipulation on application of a chemicalagent. In some embodiments, a signal generator may be capable of beingchemically destroyed on exposure to a chemical agent. Chemicaldestruction may include complete disintegration of the signal generatoror modification of the signal-generating component of the signalgenerator. Modification of the signal-generating component may includeany chemical modification (such as addition, substitution, or removal)that may result in the modification of the signal generating properties.For example, unconjugating a conjugated signal generator may result indestruction of chromogenic properties of the signal generator.Similarly, substitution of a fluorescence-inhibiting functional group ona fluorescent signal generator may result in modification of itsfluorescent properties.

In some embodiments, a signal generator may be associated with thebinder via a cleavable linker. A cleavable linker may be susceptible toa chemical agent and may dissociate, hydrolyze, or cleave when contactedwith the chemical agent. Cleavage of the cleavable linker may result inremoval of the signal generator from the binder and subsequently thebiological sample being analyzed. Suitability of a particular signalgenerator may be determined in part by the chemistry of the signalgenerator, for example, if a signal generator is amenable to destructionon application of a chemical agent then a cleavable linkage between thesignal generator and the binder may not be required. Similarly, if thesignal generator is not amenable to chemical destruction, a cleavablelinker may be used to remove the signal generator from biologicalsample. Suitable examples of signal generators are described hereinbelow.

In some embodiments, a signal generator may be selected from a lightemissive molecule, a radioisotope (e.g., P³² or H³, ¹⁴C, ¹²⁵I and ¹³¹I),an optical or electron density marker, a Raman-active tag an enzyme, anenzyme substrate (for example, a chromogenic substrate), an electronspin resonance molecule (such as for example nitroxyl radicals), anelectrical charge transferring molecule (i.e., an electrical chargetransducing molecule), a semiconductor nanocrystal, a semiconductornanoparticle, a colloid gold nanocrystal, a microbead, a magnetic bead,a paramagnetic particle, a quantum dot, or an affinity molecule (e.g., abiotin molecule, a streptavidin molecule, a protein, a peptide, nucleicacid, a carbohydrate, an antigen, a hapten, an antibody, an antibodyfragment, or a lipid).

In some embodiments, a signal generator may include a light-emissivemolecule. A light emissive molecule may emit light in response toirradiation with light of a particular wavelength. Light emissivemolecules may be capable of absorbing and emitting light throughluminescence (non-thermal emission of electromagnetic radiation by amaterial upon excitation), phosphorescence (delayed luminescence as aresult of the absorption of radiation), chemiluminescence (luminescencedue to a chemical reaction), fluorescence, or polarized fluorescence.

In some embodiments, a signal-generator may be directly detectable. Insome embodiments, a signal generator may include a chromophore. In someembodiments, a signal-generator may include a fluorescent molecule or afluorophore. Suitable chromophores and fluorophores may include one ormore molecules listed hereinabove. In some embodiments, the signalgenerator may be part of a FRET pair. FRET pair includes twofluorophores that are capable of undergoing FRET to produce or eliminatea detectable signal when positioned in proximity to one another. Someexamples of donors may include Alexa 488, Alexa 546, BODIPY 493, Oyster556, Fluor (FAM), Cy3, or TTR (Tamra). Some examples of acceptors mayinclude Cy5, Alexa 594, Alexa 647, or Oyster 656.

In some embodiments, a signal generator may essentially include afluorophore. In some embodiments, a signal generator may essentiallyinclude a fluorophore that may be attached to an antibody, for example,in an immunohistochemistry analysis. Suitable fluorophores that may beconjugated to a primary antibody include, but are not limited to,Fluorescein, Rhodamine, Texas Red, Cy2, Cy3, Cy5, VECTOR Red, ELF™(Enzyme-Labeled Fluorescence), Cy2, Cy3, Cy3.5, Cy5, Cy7, Fluor X,Calcein, Calcein-AM, CRYPTOFLUOR.TM.'S, Orange (42 kDa), Tangerine (35kDa), Gold (31 kDa), Red (42 kDa), Crimson (40 kDa), BHMP, BHDMAP,Br-Oregon, Lucifer Yellow, Alexa dye family,N-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl] (NBD), BODIPY.,boron dipyrromethene difluoride, Oregon Green, MITOTRACKER, Red,DiOC.sub.7 (3), DiIC.sub.18, Phycoerythrin, Phycobiliproteins BPE (240kDa) RPE (240 kDa) CPC (264 kDa) APC (104 kDa), Spectrum Blue, SpectrumAqua, Spectrum Green, Spectrum Gold, Spectrum Orange, Spectrum Red,NADH, NADPH, FAD, Infra-Red (IR) Dyes, Cyclic GDP-Ribose (cGDPR),Calcofluor White, Lissamine, Umbelliferone, Tyrosine or Tryptophan. Insome embodiments, a signal generator may essentially include a cyaninedye. In some embodiments, a signal generator may essentially include oneor more cyanine dye (e.g., Cy3 dye, a Cy5 dye, or a Cy7 dye).

In some embodiments, a signal generator may be indirectly detectable,for example, an enzyme/enzyme substrate combination. In someembodiments, an enzyme may precipitate a soluble substrate to form aninsoluble product (for example, in immunohistochemistry). Further, anenzyme may catalyze a chemical reaction of a chromogenic substrate thatmay be measured using a suitable technique. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, an enzyme may alter thefluorescence or chemiluminescence properties of the substrate. In someembodiments, where enzyme/enzyme substrates may be employed as signalgenerators, the enzyme-catalyzed reaction product of the substrate maybe susceptible to the chemical agent resulting in modification of theproduct (for example, color destruction using hydrogen peroxide).Suitable examples of enzyme-substrate combinations are described hereinbelow.

Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase may oxidize a dye precursor (e.g.,orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB)). Other suitable HRPO substrates may include, butare not limited to, but are not limited to, 2,2′Azino-di-3-ethylbenz-thiazoline sulfonate (ABTS, green, water soluble),aminoethyl carbazole, 3-amino, 9-ethylcarbazole AEC (3A9EC, red),Alpha-naphthol pyronin (red), 4-chloro-1-naphthol (4C1N, blue,blue-black), 3,3′-diaminobenzidine tetrahydrochloride (DAB, brown),ortho-dianisidine (green), o-phenylene diamine (OPD, brown, watersoluble), TACS Blue (blue), TACS Red (red),3,3′,5,5′Tetramethylbenzidine (TMB, green or green/blue), TRUE BLUE(blue), VECTOR VIP (purple), VECTORSG (smoky blue-gray), and Zymed BlueHRP substrate (vivid blue).

Alkaline phosphatase (AP) with para-Nitrophenyl phosphate may be used asa chromogenic substrate. Other suitable AP substrates include, but arenot limited to, AP-Blue substrate (blue precipitate, Zymed catalog p.61); AP-Orange substrate (orange, precipitate, Zymed), AP-Red substrate(red, red precipitate, Zymed), 5-bromo, 4-chloro, 3-indolyphosphate(BCIP substrate, turquoise precipitate), 5-bromo, 4-chloro, 3-indolylphosphate/nitroblue tetrazolium/iodonitrotetrazolium (BCIP/INTsubstrate, yellow-brown precipitate, Biomeda), 5-bromo, 4-chloro,3-indolyphosphate/nitroblue tetrazolium (BCIP/NBT substrate,blue/purple), 5-bromo, 4-chloro, 3-indolyl phosphate/nitrobluetetrazolium/iodonitrotetrazolium (BCIP/NBT/INT, brown precipitate, DAKO,Fast Red (Red), Magenta-phos (magenta), Naphthol AS-BI-phosphate(NABP)/Fast Red TR (Red), Naphthol AS-BI-phosphate (NABP)/New Fuchsin(Red), Naphthol AS-MX-phosphate (NAMP)/New Fuchsin (Red), New Fuchsin APsubstrate (red), p-Nitrophenyl phosphate (PNPP, Yellow, water soluble),VECTOR Black (black), VECTOR, Blue (blue), VECTOR. Red (red), Vega Red(raspberry red color), D-galactosidase (β-D-Gal) with a chromogenicsubstrate (e.g., p-nitrophenyl-β-D-galactosidase) or fluorogenicsubstrate (e.g., 4-methylumbelliferyl-β-D-galactosidase). Other suitableβ-galactosidase substrates, include, but are not limited to,5-bromo-4-chloro-3-indoyl beta-D-galactopyranoside (X-gal, blueprecipitate).

Suitable glucose oxidase (GO) substrates, include, but are not limitedto, nitroblue tetrazolium (NBT, purple precipitate), tetranitrobluetetrazolium (TNBT, black precipitate),2-(4-iodophenyl)-5-(4-nitorphenyl)-3-phenyltetrazolium chloride (INT,red or orange precipitate), Tetrazolium blue (blue), Nitrotetrazoliumviolet (violet), and3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,purple). Tetrazolium substrates may require glucose as a co-substrate.The precipitates associated with each of the substrates listedhereinabove may have unique detectable spectral signatures.

As described hereinabove, one or more of the aforementioned moleculesmay be used as a signal generator. In some embodiments, one or more ofthe aforementioned signal generators may not be amenable to chemicaldestruction and a cleavable linker may be employed to associate thesignal generator and the binder. In some embodiments, one or more of theaforementioned signal generators may be amenable to signal destructionand the signal generator may essentially include a molecule capable ofbeing destroyed chemically. In some embodiments, a signal generator mayessentially include a fluorophore capable of being destroyed chemically.In some embodiments, a signal generator may essentially include acyanine dye capable of being destroyed chemically. In some embodiments,a signal generator may essentially include one or more a Cy3 dye, a Cy5dye, or a Cy7 dye capable of being destroyed or quenched chemically.

Chemical Agents

A chemical agent may include one or chemicals capable of modifying thesignal generator or the cleavable linker (if present) between the signalgenerator and the binder. A chemical agent may be contacted with thesignal generator in the form of a solid, a solution, a gel, or asuspension. Suitable chemical agents useful to modify the signal includeagents that modify pH (for example, acids or bases), electron donors(e.g., nucleophiles), electron acceptors (e.g., electrophiles),oxidizing agents, reducing agents, or combinations thereof.

In some embodiments, a chemical agent may include a base, for example,sodium hydroxide, ammonium hydroxide, potassium carbonate, or sodiumacetate. In some embodiments, a chemical agent may include an acid, forexample, hydrochloric acid, sulfuric acid, acetic acid, formic acid,trifluoroacetic acid, or dichloroacetic acid. In some embodiments, achemical agent may include nucleophiles, for example, cyanides,phosphines, or thiols. In some embodiments, a chemical gent may includereducing agents, for example, phosphines, thiols, sodium dithionite, orhydrides that can be used in the presence of water such as borohydrideor cyanoborohydrides. In some embodiments, a chemical agent may includeoxidizing agents, for example, active oxygen species, hydroxyl radicals,singlet oxygen, hydrogen peroxide, or ozone. In some embodiments, achemical agent may include a fluoride, for example tetrabutylammoniumfluoride, pyridine-HF, or SiF₄.

One or more of the aforementioned chemical agents may be used in themethods disclosed herein depending upon the susceptibility of the signalgenerator, of the binder, of the target, or of the biological sample tothe chemical agent. In some embodiments, a chemical agent thatessentially does not affect the integrity of the binder, the target, andthe biological sample may be employed. In some embodiments, a chemicalagent that does not affect the specificity of binding between the binderand the target may be employed.

In some embodiments, where two or more (up to four) signal generatorsmay be employed simultaneously, a chemical agent may be capable ofselectively modifying one or more signal generators. Susceptibility ofdifferent signal generators to a chemical agent may depend, in part, tothe concentration of the signal generator, temperature, or pH. Forexample, two different fluorophores may have different susceptibility toa base depending upon the concentration of the base.

Sequentially Analyzing a Biological Sample, Contacting and Binding theProbe

A biological sample may be contacted with a probe to physically bind theprobe to a target in the biological sample. In some embodiments, atarget may not be easily accessible for binding the probe and abiological sample may be further processed to facilitate the bindingbetween the target and the binder (in the probe). In some embodiments, aprobe may be contacted with the biological sample in the form of asolution. Depending on the nature of the binder, the target, and thebinding between the two, sufficient contact time may be allowed. In someembodiments, an excess of binder molecules may be employed to ensure allthe targets in the biological sample are bound. After a sufficient timehas been providing for the binding action, the sample may be contactedwith a wash solution (for example an appropriate buffer solution) towash away any unbound probes. Depending on the concentration and type ofprobes used, a biological sample may be subjected to a number of washingsteps with the same or different washing solutions being employed ineach step.

In some embodiments, the biological sample may be contacted with morethan one probe in the first contacting step. The plurality of probes maybe capable of binding different targets in the biological sample. Forexample, a biological sample may include two targets: target1 andtarget2 and two sets of probes may be used in this instance: probe1(having binder1 capable of binding to target1) and probe2 (havingbinder2 capable of binding to target2). A plurality of probes may becontacted with the biological sample simultaneously (for example, as asingle mixture) or sequentially (for example, a probe1 may be contactedwith the biological sample, followed by washing step to remove anyunbound probe1, followed by contacting a probe2 with the biologicalsample, and so forth).

The number of probes that may be simultaneously bound to the target maydepend on the type of detection employed, that is, the spectralresolution achievable. For example, for fluorescence-based signalgenerators, at most four different probes (providing four spectrallyresolvable fluorescent signals) may be employed in accordance with themethods disclosed herein. Spectrally resolvable, in reference to aplurality of fluorescent signal generators, implies that the fluorescentemission bands of the signal generators are sufficiently distinct, thatis, sufficiently non-overlapping, such that, binders to which therespective signal generators are attached may be distinguished on thebasis of the fluorescent signal generated by the respective signalgenerators using standard photodetection systems. In some embodiments, abiological sample may be essentially contacted with four or less thanfour probes in the first contacting step.

In some embodiments, a biological sample may include a whole cell, atissue sample or a microarray. In some embodiments, a biological samplemay include a tissue sample. The tissue sample may be obtained by avariety of procedures including, but not limited to surgical excision,aspiration or biopsy. The tissue may be fresh or frozen. In someembodiments, the tissue sample may be fixed and embedded in paraffin.The tissue sample may be fixed or otherwise preserved by conventionalmethodology; the choice of a fixative may be determined by the purposefor which the tissue is to be histologically stained or otherwiseanalyzed. The length of fixation may depend upon the size of the tissuesample and the fixative used. For example, neutral buffered formalin,Bouin's or paraformaldehyde, may be used to fix or preserve a tissuesample.

In some embodiments, the tissue sample may be first fixed and thendehydrated through an ascending series of alcohols, infiltrated andembedded with paraffin or other sectioning media so that the tissuesample may be sectioned. In an alternative embodiment, a tissue samplemay be sectioned and subsequently fixed. In some embodiments, the tissuesample may be embedded and processed in paraffin. Examples of paraffinthat may be used include, but are not limited to, Paraplast, Broloid,and Tissuecan. Once the tissue sample is embedded, the sample may besectioned by a microtome into sections that may have a thickness in arange of from about three microns to about five microns. Once sectioned,the sections may be attached to slides using adhesives. Examples ofslide adhesives may include, but are not limited to, silane, gelatin,poly-L-lysine. In embodiments, if paraffin is used as the embeddingmaterial, the tissue sections may be deparaffinized and rehydrated inwater. The tissue sections may be deparaffinized, for example, by usingorganic agents (such as, xylenes or gradually descending series ofalcohols).

In some embodiments, aside from the sample preparation proceduresdiscussed above, the tissue section may be subjected to furthertreatment prior to, during, or following immunohistochemistry. Forexample, in some embodiments, the tissue section may be subjected toepitope retrieval methods, such as, heating of the tissue sample incitrate buffer. In some embodiments, a tissue section may be optionallysubjected to a blocking step to minimize any non-specific binding.

Following the preparation of the tissue sample, a probe solution (e.g.,labeled-antibody solution in an IHC procedure) may be contacted with thetissue section for a sufficient period of time and under conditionssuitable for binding of binder to the target (e.g., antigen in an IHCprocedure). As described earlier, two detection methods may be used:direct or indirect. In a direct detection, a signal generator-labeledprimary antibody (e.g., fluorophore-labeled primary antibody) may beincubated with an antigen in the tissue sample, which may be visualizedwithout further antibody interaction. In an indirect detection, anunconjugated primary antibody may be incubated with an antigen and thena labeled secondary antibody may bind to the primary antibody. Signalamplification may occur as several secondary antibodies may react withdifferent epitopes on the primary antibody. In embodiments where thesecondary antibody may be conjugated to an enzymatic label, achromogenic or fluorogenic substrate may be added to providevisualization of the antigen. In some embodiments two or more (at mostfour) primary antibodies (labeled or unlabeled) may be contacted withthe tissue sample. Unlabeled antibodies may be then contacted with thecorresponding labeled secondary antibodies.

Observing a First Signal from the First Probe

A signal from the signal generator in the probe may be detected using adetection system. The nature of the detection system used may dependupon the nature of the signal generators used. The detection system mayinclude an electron spin resonance (ESR) detection system, a chargecoupled device (CCD) detection system (e.g., for radioisotopes), afluorescent detection system, an electrical detection system, aphotographic film detection system, a chemiluminescent detection system,an enzyme detection system, an atomic force microscopy (AFM) detectionsystem (for detection of microbeads), a scanning tunneling microscopy(STM) detection system (for detection of microbeads), an opticaldetection system, a near field detection system, or a total internalreflection (TIR) detection system.

One or more of the aforementioned techniques may be used to observe oneor more characteristics of a first signal from a first signal generator(present in the first probe). In some embodiments, signal intensity,signal wavelength, signal location, signal frequency, or signal shiftmay be determined using one or more of the aforementioned techniques. Insome embodiments, one or more aforementioned characteristics of thesignal may be observed, measured, and recorded. In some embodiments, asignal generator may include a fluorophore and fluorescence wavelengthor fluorescent intensity may be determined using a fluorescencedetection system. In some embodiments, a signal may be observed in situ,that is, a signal may be observed directly from the signal generatorassociated through the binder to the target in the biological sample. Insome embodiments, a signal from the signal generator may be analyzedwithin the biological sample, obviating the need for separatearray-based detection systems.

In some embodiments, observing a signal may include capturing an imageof the biological sample. In some embodiments, a microscope connected toan imaging device may be used as a detection system, in accordance withthe methods disclosed herein. In some embodiments, a signal generator(such as, fluorophore) may be excited and the signal (such as,fluorescence signal) obtained may be observed and recorded in the formof a digital signal (for example, a digitalized image). The sameprocedure may be repeated for different signal generators (if present)that are bound in the sample using the appropriate fluorescence filters.Applying a Chemical Agent to Modify the First Signal

A chemical agent may be applied to the biological sample to modify thesignal. In some embodiments, signal modification may include one or moreof a change in signal characteristic, for example, a decrease inintensity of signal, a shift in the signal peak, a change in theresonant frequency, or cleavage (removal) of the signal generatorresulting in signal removal.

In some embodiments, a chemical agent may be in the form of a solutionand the biological sample may be contacted with the chemical agentsolution for a predetermined amount of time. The concentration of thechemical agent solution and the contact time may be dependent on thetype of signal modification desired. In some embodiments, the contactingconditions for the chemical agent may be selected such that the binder,the target, the biological sample, and binding between the binder andthe target may not be affected. In some embodiments, a chemical agentmay only affect the signal generator and the chemical agent may notaffect the target/binder binding or the binder integrity. Thus by way ofexample, a binder may include a primary antibody or a primaryantibody/secondary combination. A chemical agent according to themethods disclosed herein may only affect the signal generator, and theprimary antibody or primary antibody/secondary antibody combination mayessentially remain unaffected. In some embodiments, a binder (such as, aprimary antibody or primary antibody/secondary antibody combination) mayremain bound to the target in the biological sample after contacting thesample with the chemical agent. In some embodiments, a binder may remainbound to the target in the biological sample after contacting the samplewith the chemical agent and the binder integrity may remain essentiallyunaffected (for example, an antibody may not substantially denature orelute in the presence of a chemical agent).

In some embodiments, a characteristic of the signal may be observedafter contacting the sample with a chemical agent to determine theeffectiveness of the signal modification. For example, a color may beobserved before application of the chemical agent and the color may beabsent after application of the chemical agent. In another example,fluorescence intensity from a fluorescent signal generator may beobserved before contacting with the chemical agent and after contactingwith the chemical agent. In some embodiments, a decrease in signalintensity by a predetermined amount may be referred to as signalmodification. In some embodiments, modification of the signal may referto a decrease in the signal intensity by an amount in a range of greaterthan about 50 percent. In some embodiments, modification of the signalmay refer to a decrease in the signal intensity by an amount in a rangeof greater than about 60 percent. In some embodiments, modification ofthe signal may refer to a decrease in the signal intensity by an amountin a range of greater than about 80 percent.

Contacting the Sample with a Subsequent Probe and Binding the to aSubsequent Target

The biological sample may be contacted with a second probe using one ormore procedures described herein above for the first probe. The secondprobe may be capable of binding to target different from the targetbound by the first probe. In embodiments where a plurality of probes maybe contacted with the biological sample in the first probe contact step,the second probe may be capable of binding a target different from thetargets bound by the first probe set. In some embodiments, a biologicalsample may be contacted with a plurality of probes in the second probecontact step.

Observing a Second Signal from a Subsequent Probe.

One or more detection methods described hereinabove may be used toobserve one or more characteristics of a subsequent (e.g., second,third, etc.) signal from a second signal generator (present in thesubsequent probe). In some embodiments, signal intensity, signalwavelength, signal location, signal frequency, or signal shift may bedetermined using one or more of the aforementioned techniques. Similarto the first signal, a subsequent signal (for example, a fluorescencesignal) obtained may be recorded in the form of a digital signal (forexample, a digitalized image). In some embodiments, observing asubsequent signal may also include capturing an optical image of thebiological sample.

Contacting the Sample with One or More Morphological Stain

In some embodiments, a biological sample may include a cell or a tissue,and the sample may be contacted with a morphological stain before,during, or after the contacting step with the first probe or secondprobe. A morphological stain may include a dye that may stain differentcellular components, in order to facilitate identification of cell typeor disease status. In some embodiments, the morphological stain may bereadily distinguishable from the signal generators in the first probeand the second probe, that is, the stain may not emit signal that mayoverlap with signal from the first probe or the second probe. Forexample, for a fluorescent morphological stain, the signal from themorphological stain may not auto fluoresce in the same wavelength as thefluorophores used in the first probe or the second probe.

A morphological stain may be contacted with the biological samplebefore, during, or after, any one of the aforementioned steps. In someembodiments, a morphological stain may be contacted with biologicalsample along with the first probe. In some embodiments, a morphologicalstain may be contacted with the biological sample before contacting thesample with a chemical agent and after physically binding the firstprobe to the target. In some embodiments, a morphological stain may becontacted with a biological sample after contacting the sample with achemical agent and modifying the signal. In some embodiments, amorphological stain may be contacted with a biological sample along withthe second probe. In some embodiments, a biological sample may becontacted with the morphological stain after binding the second probe tothe target. In some embodiments, a morphological stain may be stabletowards a chemical agent, that is, the signal generating properties ofthe morphological stain may no be substantially affected by the chemicalagent. In some embodiments, where a biological sample may be stainedwith a probe and a morphological stain at the same time, application ofchemical agent to modify the signal from the probe may not modify thesignal from the morphological stain.

In some embodiments, chromophores, fluorophores, or enzyme/enzymesubstrates may be used as morphological stains. Suitable examples ofchromophores that may be used as morphological stains (and their targetcells, subcellular compartments, or cellular components) may include,but are not limited to, but are not limited to, Eosin (alkaline cellularcomponents, cytoplasm), Hematoxylin (nucleic acids), Orange G (redblood, pancreas, and pituitary cells), Light Green SF (collagen),Romanowsky-Giemsa (overall cell morphology), May-Grunwald (blood cells),Blue Counterstain (Trevigen), Ethyl Green (CAS) (amyloid),Feulgen-Naphthol Yellow S (DNA), Giemsa (differentially stains variouscellular compartments), Methyl Green (amyloid), pyronin (nucleic acids),Naphthol-Yellow (red blood cells), Neutral Red (nuclei), Papanicolaoustain (a mixture of Hematoxylin, Eosin Y, Orange G and Bismarck Brownmixture (overall cell morphology)), Red Counterstain B (Trevigen), RedCounterstain C (Trevigen), Sirius Red (amyloid), Feulgen reagent(pararosanilin) (DNA), Gallocyanin chrom-alum (DNA), Gallocyaninchrom-alum and Naphthol Yellow S (DNA), Methyl Green-Pyronin Y (DNA),Thionin-Feulgen reagent (DNA), Acridine Orange (DNA), Methylene Blue(RNA and DNA), Toluidine Blue (RNA and DNA), Alcian blue(carbohydrates), Ruthenium Red (carbohydrates), Sudan Black (lipids),Sudan IV (lipids), Oil Red-O (lipids), Van Gieson's trichrome stain(acid fuchsin and picric acid mixture) (muscle cells), Masson trichromestain (hematoxylin, acid fuchsin, and Light Green mixture) (stainscollagen, cytoplasm, nucleioli differently), Aldehyde Fuchsin (elastinfibers), or Weigert stain (differentiates reticular and collagenousfibers).

Examples of suitable fluorescent morphological stains (and their targetcells, subcellular compartments, or cellular components if applicable)may include, but are not limited to: 4′,6-diamidino-2-phenylindole(DAPI) (nucleic acids), Eosin (alkaline cellular components, cytoplasm),Hoechst 33258 and Hoechst 33342 (two bisbenzimides) (nucleic acids),Propidium Iodide (nucleic acids), Spectrum Orange (nucleic acids),Spectrum Green (nucleic acids), Quinacrine (nucleic acids),Fluorescein-phalloidin (actin fibers), Chromomycin A 3 (nucleic acids),Acriflavine-Feulgen reaction (nucleic acid), Auramine O-Feulgen reaction(nucleic acids), Ethidium Bromide (nucleic acids). Niss1 stains(neurons), high affinity DNA fluorophores such as POPO, BOBO, YOYO andTOTO and others, and Green Fluorescent Protein fused to DNA bindingprotein (e.g., histones), ACMA, Quinacrine and Acridine Orange.

Examples of suitable enzymes (and their primary cellular locations oractivities) may include, but are not limited to, ATPases (musclefibers), succinate dehydrogenases (mitochondria), cytochrome c oxidases(mitochondria), phosphorylases (mitochondria), phosphofructokinases(mitochondria), acetyl cholinesterases (nerve cells), lactases (smallintestine), acid phosphatases (lysosomes), leucine aminopeptidases(liver cells), dehydrogenases (mitochondria), myodenylate deaminases(muscle cells), NADH diaphorases (erythrocytes), and sucrases (smallintestine).

Reiteration of the Contacting, Binding, and Observing Steps

In some embodiments, after contacting the sample with a subsequent(e.g., second, third, etc.) probe, agent modification and subsequentprobe administration may be repeated multiple times. In someembodiments, after observing a second signal from the second probe, thebiological sample may be contacted with a chemical agent to modify thesignal from the second probe. Furthermore, a third probe may becontacted with the biological sample, wherein the third probe may becapable of binding a target different from the first and the secondprobes. Likewise, a signal from the third probe may be observed followedby application of chemical agent to modify the signal. The contacting,binding, and observing steps may be repeated iteratively multiple timesusing an n^(th) probe capable of binding to additional targets toprovide the user with information about a variety of targets using avariety of probes and/or signal generators.

In some embodiments, a series of probes may be contacted with thebiological sample in a sequential manner to obtained a multiplexedanalysis of the biological sample. In some embodiments, a series ofprobe sets (including at most 4 probes in one set) may be contacted withthe biological sample in a sequential manner to obtained a multiplexedanalysis of the biological sample. Multiplexed analysis generally refersto analysis of multiple targets in a biological sample using the samedetection mechanism.

Correlating the First Signal and the Subsequent Signals

In some embodiments, a first signal, a second signal, or both firstsignal and the second signal may be analyzed to obtain informationregarding the biological sample. For example, in some embodiments, apresence or absence of a first signal may indicate the presence orabsence of the first target (capable of binding to the first binder) inthe biological sample. Similarly, the presence or absence of a secondsignal may indicate the presence or absence of the second target(capable of binding to the second binder in the biological sample). Inembodiments where multiple targets may be analyzed using a plurality ofprobes, the presence or absence of a particular signal may indicate thepresence or absence of corresponding target in the biological sample.

In some embodiments, an intensity value of a signal (for example,fluorescence intensity) may be measured and may be correlated to theamount of target in the biological sample. A correlation between theamount of target and the signal intensity may be determined usingcalibration standards. In some embodiment, intensity values of the firstand second signals may be measured and correlated to the respectivetarget amounts. In some embodiments, by comparing the two signalintensities, the relative amounts of the first target and the secondtarget (with respect to each other or with respect to a control) may beascertained. Similarly, where multiple targets may be analyzed usingmultiple probes, relative amounts of different targets in the biologicalsample may be determined by measuring different signal intensities. Insome embodiments, one or more control samples may be used as describedhereinabove. By observing a presence or absence of a signal in thesamples (biological sample of interest versus a control), informationregarding the biological sample may be obtained. For example bycomparing a diseased tissue sample versus a normal tissue sample,information regarding the targets present in the diseased tissue samplemay be obtained. Similarly by comparing signal intensities between thesamples (i.e., sample of interest and one or more control), informationregarding the expression of targets in the sample may be obtained.

In some embodiments, a location of the signal in the biological samplemay be observed. In some embodiments, a localization of the signal inthe biological signal may be observed using morphological stains. Insome embodiments relative locations of two or more signals may beobserved. A location of the signal may be correlated to a location ofthe target in the biological sample, providing information regardinglocalization of different targets in the biological sample. In someembodiments, an intensity value of the signal and a location of thesignal may be correlated to obtain information regarding localization ofdifferent targets in the biological sample. For examples certain targetsmay be expressed more in the cytoplasm relative to the nucleus, or viceversa. In some embodiments, information regarding relative localizationof targets may be obtained by comparing location and intensity values oftwo or more signals.

In some embodiments, one or more of the observing or correlating stepmay be performed using computer-aided means. In embodiments where thesignal(s) from the signal generator may be stored in the form of digitalimage(s), computer-aided analysis of the image(s) may be conducted. Insome embodiments, images (e.g., signals from the probe(s) andmorphological stains) may be overlaid using computer-aidedsuperimposition to obtain complete information of the biological sample,for example topological and correlation information.

In some embodiments, one or more of the aforementioned steps (a) to (g)may be automated and may be performed using automated systems. In someembodiments, all the steps (a) to (g) may be performed using automatedsystems.

The methods disclosed herein may find applications in analytic,diagnostic, and therapeutic applications in biology and in medicine. Insome embodiments, the methods disclosed herein may find applications inhistochemistry, particularly, immunohistochemistry. Analysis of cell ortissue samples from a patient, according to the methods describedherein, may be employed diagnostically (e.g., to identify patients whohave a particular disease, have been exposed to a particular toxin orare responding well to a particular therapeutic or organ transplant) andprognostically (e.g., to identify patients who are likely to develop aparticular disease, respond well to a particular therapeutic or beaccepting of a particular organ transplant). The methods disclosedherein, may facilitate accurate and reliable analysis of a plurality(e.g., potentially infinite number) of targets (e.g., disease markers)from the same biologically sample.

EXAMPLES

The following examples are intended only to illustrate methods andembodiments in accordance with the invention, and as such should not beconstrued as imposing limitations upon the claims.

The following examples 1-6 illustrate embodiments in which labels arechemically destroyed by oxidizing agents, nucleophiles or changes in pH.Oxidizing agents used are hydrogen peroxide (H₂O₂) and sodium periodate(NaIO₄). Changes in pH are obtained using a base like sodium hydroxide(NaOH). Tris(2-carboxyethyl)phosphate created in situ by dissolvingTCEP.HCl in base is a nucleophile. Labels include cyanine dyes, such as,Cy3, Cy5, and Cy7 and nuclear labels, such as,4′,6-diamidino-2-phenylindole (DAPI). Selective destruction of labelsmay be obtained by varying the concentration, type, and time of exposureto the oxidizing agent or base.

Example 1 Selective Oxidation using Hydrogen Peroxide of Cyanine Dyeswithout Affecting DAPI

A solution of hydrogen peroxide (H₂O₂) was prepared in a sodiumbicarbonate buffer by mixing 1 volume of 1 molar (1M) sodiumbiocarbonate, 3 volumes of water, and 1 volume of 30 percent (v/v)hydrogen peroxide. pH of sodium bicarbonate was adjusted to a pH 10using sodium hydroxide, prior to mixing with hydrogen peroxide.

Three separate solutions of cyanine dyes: Cy3, Cy5, and Cy7 wereprepared in water at a concentration of about 2 micromolar (2 μM). Analiquot of a cyanine dye solution was mixed with an aliquot of the H₂O₂solution to prepare a sample solution with a final concentration ofabout 3 volume percent H₂O₂ and 1 micromolar (1 μM) cyanine dye (Samples1 (Cy3), 2 (Cy5), and 3 (Cy7)). A 1 micromolar (1 μM) cyanine dyesolution in water (without H₂O₂) was used as a control.

Oxidation reaction of the cyanine dye was monitored by measuringabsorbance spectrum of the dye on an ultraviolet/visible (UV/Vis)spectrophotometer as a function of time. FIG. 1 shows the absorbancespectra of Sample 1 as a function of wavelength, after duration of 10minutes and 15 minutes. The absorbance value decreased considerably whencompared with the control. FIG. 2 shows the absorbance values forSamples 1, 2, and 3 as a function of wavelength. The absorbance of theSamples 1, 2 and 3 reduced to zero after a duration of time exhibitingchemical destruction of the dye by H₂O₂. The time duration for theSamples 1, 2 and 3 was different for the different dye: 19 minutes forSample 1, 15 minutes for Sample 2, and 3 minutes for Sample 3.

A solution of 4′,6-diamidino-2-phenylindole (DAPI) was prepared in waterat a concentration of about 57 μM. An aliquot of DAPI solution was mixedwith an aliquot of the H₂O₂ solution to prepare a solution with a finalconcentration of about 3 volume percent H₂O₂ and 10micrograms/milliliters DAPI (Sample 4). A 10 micrograms/milliliters DAPIsolution in water (without H₂O₂) was used as a control.

Oxidation reaction of DAPI was monitored by measuring absorbancespectrum of Sample 4 as a function of time. FIG. 3 shows the absorbancespectra of Sample 4 as a function of wavelength, after duration of 30minutes and 140 minutes. The absorbance value of Sample 4 did not varymuch even after a period of 140 minutes and was in the same range as thecontrol, exhibiting no significant effect of H₂O₂ on DAPI.

Example 2 Selective Oxidation (Using Sodium Periodate) of Cyanine Dyeswithout Affecting DAPI

A solution of sodium periodate (NaIO₄) was prepared by mixing a 0.2molar solution of NaIO₄ in 0.1× phosphate buffer saline (PBS). Threeseparate solutions of cyanine dyes, Cy3, Cy5, and Cy7 were prepared inwater at a concentration of about 2 micromolar (2 μM). An aliquot of acyanine dye solution was mixed with an aliquot of the NaIO₄ solution toprepare a solution with a final concentration of about 0.1 molar (1 μM)NaIO₄ and 1 micromolar (1 μM) cyanine dye (Samples 5 (Cy3), 6 (Cy5), and7 (Cy7)). A 1 micromolar (1 μM) cyanine dye solution in water (withoutNaIO₄) was used as a control.

Oxidation reaction of the cyanine dye was monitored by measuringabsorbance spectrum of the dye on an ultraviolet/visible (UV/Vis)spectrophotometer as a function of time. FIG. 4 shows the absorbancespectra of Sample 5 as a function of wavelength, after duration of 20minutes, 60 minutes and 210 minutes. The absorbance value decreased asfunction of time when compared with the control. Complete absorbanceloss was observed after 210 minutes for Sample 5. FIG. 5 shows theabsorbance spectra of Sample 6 as a function of wavelength, afterduration of 12 minutes and 16 minutes. The absorbance value decreased asfunction of time when compared with the control. Complete absorbanceloss was observed rapidly, after 16 minutes.

A solution of 4′,6-diamidino-2-phenylindole (DAPI) was prepared in waterat a concentration of about 57 μM. An aliquot of DAPI solution was mixedwith an aliquot of the NaIO₄ solution to prepare a solution with a finalconcentration of about 0.1M NaIO₄ and 10 micrograms/milliliters DAPI(Sample 8). A 10 micrograms/milliliters DAPI solution in water (withoutNaIO₄) was used as a control.

Oxidation reaction of DAPI was monitored by measuring absorbancespectrum of Sample 8 as a function of time. FIG. 6 shows the absorbancespectra of Sample 8 as a function of wavelength, after duration of 22minutes, 70 minutes and 210 minutes. The absorbance value of Sample 8did not vary much even after a period of 210 minutes and a significantamount of DAPI remained intact when compared to the control.

Example 3 Selective Destruction (Using Sodium Hydroxide Base) of CyanineDyes without Affecting DAPI

A NaOH solution was prepared at a concentration of 0.1M and 1M. Threeseparate solutions of cyanine dyes, Cy3, Cy5, and Cy7 were prepared inwater at a concentration of 2 μM. An aliquot of a cyanine dye solutionwas mixed with an aliquot of the NaOH solution of two differentconcentrations of NaOH: 0.1M NaOH (Samples 9a, 10a, and 11a) and 1M NaOH(Samples 9b, 10b, and 11b). A 1 μM cyanine dye solution in water(without NaOH) was used as a control.

Base destruction of the dye was monitored by measuring absorbancespectrum of the samples as a function of time. FIG. 7 shows theabsorbance spectra of Samples 9a, 10a, and 11a as a function ofwavelength, after duration of less than 5 minutes. The absorbance valueof Sample 9a did not vary much and a significant amount of Cy3 remainedintact when compared to the control. Sample 10a showed a 20 percentdecomposition of the Cy5 dye, while Sample 11a showed a 70 percentdecomposition of the Cy7 dye using 0.1 M NaOH. FIG. 8 shows theabsorbance spectra of Samples 9b and 10b as a function of wavelength,after duration of less than 5 minutes. Sample 9b showed a 50 percentdecomposition of the Cy3 dye, while Sample 10b showed an 80 percentdecomposition of the Cy5 dye using 1 M NaOH.

A solution of 4′,6-diamidino-2-phenylindole (DAPI) was prepared in waterat a concentration of about 57 μM. An aliquot of DAPI solution was mixedwith an aliquot of the NaOH solution of two different concentrations0.1M NaOH (Samples 12a) and 1M NaOH (Samples 12b). A 10micrograms/milliliters DAPI dye solution in water (without NaOH) wasused as a control. Base destruction of DAPI was monitored by measuringabsorbance spectrum of Samples 12a and 12b as a function of time. FIG. 9shows the absorbance spectra of Samples 12a and 12b as a function ofwavelength. The absorbance value of the samples did not vary much and asignificant amount of DAPI remained intact when compared to the control.

Example 4 Selective Destruction of Cy5 and Cy7 Dyes without AffectingCy3

A 5 percent (w/v) solution of a nucleophile was prepared by mixingtris[2-carboxyethyl]-phosphine hydrochloride (TCEP.HCl) in 1M sodiumbiocarbonate buffer (final pH 7.7). Three separate solutions of cyaninedyes, Cy3, Cy5, and Cy7 were prepared in water at a concentration ofabout 2 μM. An aliquot of a cyanine dye solution was mixed with analiquot of the TCEP.HCl solution to prepare Samples 13 (Cy3), 14 (Cy5),and 15 (Cy7). A 1 μM cyanine dye solution in water (without TCEP.HCl)was used as a control.

Destruction of the dye was monitored by measuring absorbance spectrum ofthe samples as a function of time. FIG. 10 shows the absorbance spectraof Samples 13, 14, and 15 as a function of wavelength. The absorbancevalue of Sample 13 did not vary much and a significant amount of Cy3remained intact when compared to the control after duration of 60minutes. Samples 14 and 15 showed a significant decomposition of the Cy5and Cy7 dyes after duration of 0.5 minutes.

Example 5 Selective Destruction of Cy7 Dye without Affecting Cy3

A solution of hydrogen peroxide (H₂O₂) was prepared in a phosphatebuffer saline solution (PBS) by mixing a 6 percent (v/v) solution ofH2O2 in 0.8× PBS (final pH 6.6). Two separate solutions of cyanine dyes,Cy3 and Cy7 were prepared in water at a concentration of about 2micromolar (2 μM). An aliquot of a cyanine dye solution was mixed withan aliquot of the H₂O₂ solution to prepare Samples 16 (Cy3) and 17(Cy7)). A 1 micromolar (1 μM) cyanine dye solution in water (withoutH₂O₂) was used as a control.

Destruction of the dye was monitored by measuring absorbance spectrum ofthe samples as a function of time. FIG. 11 shows the absorbance spectraof Samples 16 and 17 as a function of wavelength. The absorbance valueof Sample 16 did not vary much and a significant amount of Cy3 remainedintact when compared to the control after duration of 60 minutes. Sample17 showed a significant decomposition of the Cy7 dye after duration of60 minutes.

The following examples 7-21 illustrate embodiments of the inventionaccording to which multiple imaging of tissue samples is conducted.Multiple staining is obtained by staining, imaging, chemicallydestroying the stain, restaining, imaging, and repeating the steps.

Example 6 Preparation of Tissue Samples for Staining

Adult human tissue samples were obtained as tissue slides embedded inparaffin. The tissue samples included slides of colon (Biochain,T2234090), normal breast tissue (Biochain, T2234086), prostate cancer(Biochain, T2235201-1), colon adenocarcinoma (Biochain, T2235090-1),breast tissue microarray (Imgenex, IMH 367, p61), breast TMA (ImegenexIMH 367, p32), and normal prostrate (Biochain, T2234201). Paraffinembedded slides of adult human tissue were subjected to animmunohistochemistry protocol to prepare them for staining. The protocolincluded deparaffinization, rehydration, incubation, and wash.Deparaffinization was carried by washing the slides with Histochoice (ortoluene) for a period of 10 minutes and with frequent agitation. Afterdeparaffinization, the tissue sample was rehydrated by washing the slidewith ethanol solution. Washing was carried out with three differentsolutions of ethanol with decreasing concentrations. The concentrationsof ethanol used were 90 volume percent, 70 volume percent, and 50 volumepercent. The slide was then washed with a phosphate buffer saline (PBS,pH 7.4). Membrane permeabilization of the tissue was carried out bywashing the slide with 0.1 weight percent solution of Triton TX-100.Citrate buffer pH 6.0 (Vector Unmasking Solution) was used for antigenretrieval. The slides were exposed to the buffer in a pressure cookerfor a period of 15 minutes followed by cooling at room temperature for aperiod of 20 minutes. The slide was then blocked against nonspecificbinding by washing with PBS and 900 microliters of 3 volume percentbovine serum albumin (BSA) for 45 minutes at 37 degrees Celsius. Forstaining with secondary antibodies (optional), the slide was alsoblocked with 100 microliters of serum from secondary antibody hostspecies.

Example 7 Conjugation of Antibodies with a Dye

Dye-conjugated antibodies were prepared according to the followingprocedure. The antibodies used for conjugating and staining includedanti proliferating cell nuclear antigen, clone pc10 (Sigma Aldrich,P8825); anti smooth muscle alpha actin (SmA), clone 1A4 (Sigma, A2547);rabbit anti beta catenin (Sigma, C 2206); mouse anti pan cytokeratin,clone PCK-26 (Sigma, C1801); mouse anti estrogen receptor alpha, clone1D5 (DAKO, M 7047); beta catenin antibody, clone 15B8 (Sigma, C 7738);goat anti vimentin (Sigma, V4630); cycle androgen receptor clone AR441(DAKO, M3562); Von willebrand factor 7, keratin 5, keratin 8/18,e-cadherin, Her2/neu, Estrogen receptor, p53, progesterone receptor,beta catenin; donkey anti-mouse (Jackson Immunoresearch, 715-166-150);and donkey anti rabbit (Jackson Immunoresearch, 711-166-152).

A micron YM-10 spin column was wetted with 250 microliters of PBS andthe column was spun for 15 minutes. 500 milliliters of the antibody (200micrograms/milliliters) was pipetted into the wet column. The column wasspun for 30 minutes at 11000 rpm at 4 degrees Celsius. The concentratedantibody/protein was then transferred into a new tube and spun for 30seconds to remove the concentrated protein. A coupling buffer solutionwas then mixed with the concentrated antibody solution. The couplingbuffer solution included 1M sodium carbonate (pH between 8-9) and 5microliters of the buffer was used per 100 microliters of the antibodysolution. The antibody and buffer solution was added to 0.01-0.1milligrams of the cyanine dye. The dye was reconstituted in DMSO to a10-20-milligrams/milliliter concentration prior to incubating with theantibody. The resulting solution was mixed thoroughly by pipetting andany bubbles formed were removed by spinning the tube. The solution wascovered with a foil and incubated at room temperature for a period ofabout 30-45 minutes. Post incubation the solution was added to YM-10spin column and spun for 30 minutes at 4 degrees Celsius at 11000 rpm.The solution was washed with PBS and spun to remove any unconjugated dyeor antibody. The dye-conjugated antibody solution was then diluted with50 percent glycerol and stored in a freezer at −20 degrees Celsius.

Example 8 Staining and Imaging of Tissue with Dyes

A slide prepared in Example 6 was incubated with a dye-conjugatedantibody prepared in Example 7. Incubation was conducted in 3 percentBSA for 45 minutes at 37 degrees Celsius. After incubation, the slidewas subjected to an extensive series of PBS washes. When secondaryantibodies (optional) were used, the slide was incubated with asecondary antibody in BSA for 45 minutes at 37 degrees Celsius. Afterincubation, the slide was subjected to an extensive series of PBSwashes. A primary antibody or secondary antibody (optional)-stainedslide was optionally counterstained with the morphological stain, DAPI,and cover slipped.

A cover slipped slide was imaged using a camera. The camera used was amonochromatic Leica DFC 350FX monochromatic high-resolution cameramounted in a Leica DMRA2 fluorescent microscope. The magnification usedwas 20× unless otherwise stated. After image acquisition, the cover slipwas removed and the slide was washed with PBS to prepare for signaldestruction.

Example 9 Dye Destruction, Staining and Imaging

NaOH solution and H₂O₂ solution were used for signal destruction. A NaOHsolution was prepared suing 500 microliters of 50 volume percent NaOHand 49.5 milliliters of PBS. The final pH of the NaOH solution wasaround 11.9-12.5. A H₂O₂ solution was prepared by mixing 10 millilitersof 0.5M sodium carbonate (pH 10), 5 milliliters of 30 volume percentH₂O₂, and 35 milliliters of water. A slide was placed in the NaOH orH₂O₂ solution for 15 minutes with gentle agitation. After 15 minutes,the slide was washed again with PBS, cover slipped and either imagedagain (optional) to check the efficacy of the dye destruction orrestained and imaged. Restaining and reimaging steps were carried outusing the process described in Example 8. Following imaging, a slide wassubjected to signal destruction, staining, and imaging cycles, and theprocess was repeated a multiple number of times. The tissue samples wereimaged using 1-9 different antibodies. After imaging with the cyanineseries, the slide was optionally stained and imaged with morphologicalstains H&E.

Example 10 Single Channel Staining and Imaging of a Normal Colon Tissueusing NaOH

A normal colon slide was stained with a primary antibody mouse antiproliferating cell nuclear antigen (PCNA) clone pc 10, and targeted witha Cy3-conjugated donkey anti-mouse to form Sample 18a. Sample 18a wasimaged and then treated with a NaOH solution to form Sample 18b, whichwas imaged again. Staining, imaging, and dye destruction steps wereperformed according to the procedures described hereinabove in Examples8 and 9. FIG. 12 shows micrographs (at 10× magnification) of Sample 18a(before dye destruction) and Sample 18b (after dye destruction). Aftertreatment with NaOH little or no signal from Cy3 remained.

Example 11 Single Channel Staining and Imaging of a Normal Colon Tissueusing NaOH

A normal colon slide was stained with a primary antibody mouse antismooth muscle alpha actin (SmA) clone 1A4, and targeted with aCy3-conjugated donkey anti-mouse to form Sample 19a. Sample 19a wasimaged and then treated with a NaOH solution to form Sample 19b, whichwas imaged again. Staining, imaging, and dye destruction steps wereperformed according to the procedures described hereinabove in Examples8 and 9. FIG. 13 shows micrographs (at 10× magnification) of Sample 19a(before dye destruction) and Sample 19b (after dye destruction). Aftertreatment with NaOH a little amount of signal from Cy3 remained.

Example 12 Two Channel Staining and Imaging of a Normal Breast Tissueusing NaOH

A normal breast tissue was stained with a primary antibody SmA, targetedwith a Cy3-conjugated donkey anti-mouse, and counter-stained with DAPIto form Sample 20a. Sample 20a was imaged and then treated with a NaOHsolution to form Sample 20b, which was imaged again. Staining, imaging,and dye destruction steps were performed according to the proceduresdescribed hereinabove in Examples 8 and 9. Sample 20b was restained witha primary antibody rabbit anti beta catenin, and targeted with aCy3-conjugated anti-rabbit to form Sample 20c. Sample 20c was imaged andthen counter-stained with H&E to form Sample 20d and imaged again.

FIG. 14 shows micrographs of Sample 20a (before dye destruction) andSample 20b (after dye destruction). After treatment with NaOH little orno signal from Cy3 remained and only DAPI was observed. Micrograph ofSample 20c showed imaging in the same Cy3 channel was possible bystaining with a different antibody. Morphological information about thetissue was obtained by further staining with H&E (Sample 20d).

Example 13 Two Channel Staining and Imaging of a Prostrate Cancer Tissueusing NaOH

A prostrate cancer tissue was stained with a primary antibody mouse antipan cytokeratin clone PCK-26, and targeted with a Cy3-conjugated donkeyanti-mouse, to form Sample 21a. Sample 21a was imaged and thencounterstained with DAPI to form Sample 21b. Sample 21b was imaged andthen treated with a NaOH solution to form Sample 21c, which was imagedagain. Staining, imaging, and dye destruction steps were performedaccording to the procedures described hereinabove in Examples 8 and 9.Sample 21c was restained with a primary antibody SmA, and targeted witha Cy3-conjugated anti-rabbit to form Sample 21d and imaged again.

FIG. 15 shows micrographs of Samples 21a and b (before dye destruction)and Sample 21c (after dye destruction). After treatment with NaOH littleor no signal from Cy3 remained and only DAPI was observed. Micrograph ofSample 21d showed imaging in the same Cy3 channel was possible bystaining with a different antibody. Nuclear information about the tissuewas obtained by staining with DAPI (Sample 21b).

Example 14 Two Channel Staining and Imaging of a Colon Adenocarcinomausing NaOH

A colon adenocarcinoma slide was stained with a primary antibody mouseanti pan cytokeratin clone PCK-26, and targeted with a Cy3-conjugateddonkey anti-mouse, to form Sample 22a. Sample 22a was imaged and thencounterstained with DAPI to form Sample 22b. Sample 22b was imaged andthen treated with a NaOH solution to form Sample 22c, which was imagedagain. Staining, imaging, and dye destruction steps were performedaccording to the procedures described hereinabove in Examples 8 and 9.Sample 22c was restained with a primary antibody SmA, and targeted witha Cy3-conjugated anti-rabbit to form Sample 22d and imaged again.

FIG. 16 shows micrographs of Samples 22a and b (before dye destruction)and Sample 22c (after dye destruction). After treatment with NaOH littleor no signal from Cy3 remained and only DAPI was observed. Micrograph ofSample 22d showed imaging in the same Cy3 channel was possible bystaining with a different antibody. Nuclear information about the tissuewas obtained by staining with DAPI (Sample 22b).

Example 15 Two Channel Staining and Imaging of a Breast TissueMicroarray with Baseline Measurement using NaOH

A breast tissue microarray (Sample 23a) was imaged in the DAPI and Cy3channel to measure the autofluorescence from the tissue. Sample 23a wasthen stained with DAPI to form Sample 23b, imaged and then treated withNaOH to form Sample 23c, and imaged again. Sample 23a was also stainedwith a primary antibody mouse anti estrogen receptor alpha clone 1D5,and targeted with a Cy3-conjugated donkey anti-mouse, to form Sample23d. Sample 23d was imaged then treated with a NaOH solution to formSample 23e, which was imaged again. Staining, imaging, and dyedestruction steps were performed according to the procedures describedhereinabove in Examples 8 and 9.

FIG. 17 shows micrographs of Samples 23a-e. Micrographs of Samples 23cand 23e were compared to the autofluorescence (baseline) observed inSample 23a. Both Samples showed signal reduction. DAPI-stained sampleshowed signal reduction possibly due to destruction of nucleic acids towhich DAPI binds.

Example 16 Three Channel Staining and Imaging of Breast TMA using NaOH

A breast TMA slide was stained with a primary antibody mouse anti pancytokeratin clone PCK-26, targeted with a Cy3-conjugated donkeyanti-mouse, and counterstained with DAPI to form Sample 24a. Sample 24awas imaged and then treated with a NaOH solution to form Sample 24b,which was imaged again. Staining, imaging, and dye destruction stepswere performed according to the procedures described hereinabove inExamples 8 and 9. Sample 24b was restained with a Cy3-directlyconjugated beta catenin antibody to form Sample 24c and imaged again.The array was again treated with NaOH and labeled with Cy3-directconjugated SmA antibody to form Sample 24d and imaged again. The imagesobtained were registered, pseudo colored and overlaid (Sample 24e) togive spatial information for expressing antigen. Sample 24d was furtherstained with H&E to form Sample 24f.

FIG. 18 shows micrographs of Sample 24a (before dye destruction) andSample 24b (after dye destruction). After treatment with NaOH little orno signal from Cy3 remained and only DAPI was observed. Micrographs ofSamples 24c and 24d showed imaging in the same Cy3 channel was possibleby staining with different antibodies. Morphological information aboutthe tissue was obtained by staining with H&E (Sample 24f).

Example 17 Twelve Channel Staining and Imaging of Normal Prostrate usingNaOH

Images were taken prior to staining to baseline the autofluorescencecoming from each channel. A normal prostrate slide was stained with acocktail of primary antibodies: mouse anti pan cytokeratin clone andgoat anti vimentin. The two primary antibodies were targeted with asecond cocktail of secondary antibodies Cy3-conjugated donkey anti-mouseand Cy5-conjugated donkey anti-goat to form Sample 25a. Sample 25a wasimaged and then treated with a NaOH solution. The tissue was thenstained with a second cocktail of primary antibodies: androgen receptorclone and alpha catenin. The two primary antibodies were targeted withanother cocktail of Cy-3 and Cy-5 conjugated secondary antibodies toform Sample 25b. Sample 25b was imaged and then treated with a NaOHsolution. This was followed by staining-imaging-NaOH treatment-stainingsteps using seven Cy-directly conjugated antibodies (Samples 25c-25i).The antibodies used were: smooth muscle alpha actin, beta catenin, pancadherin, Von willebrand factor 7, keratin 5, keratin 8/18, ande-cadherin. Each staining step included counterstaining with DAPI(Sample 25j).

FIG. 19 shows micrographs of Sample 25a (Cy3 and Cy5 channels), Sample25b (Cy3 and Cy5 channels), and Samples 25c-25j. FIG. 19 shows thatmultiple imaging in the same Cy3 channel was possible by staining withdifferent antibodies. 12-channel multiple imaging was possible with 9 ofthe channels being Cy3 channels.

Example 18 Four Channel Staining and Imaging of Normal Prostrate usingH₂O₂

Images were taken prior to staining to baseline the autofluorescencecoming from each channel. A normal prostrate slide was stained with aCy3-directly conjugated pan cadherin to form Sample 26a. The slide wasimaged and treated with H2O2 (Sample 26b), restained with Cy3-conjugatedSmA (Sample 26c), treated with H2O2 (Sample 26d), restained withCy3-conjugated pan cytokeratin (Sample 26e), treated with H₂O₂ (Sample26f), restained with Cy-conjugated vimentin (Sample 26g), and treatedwith H₂O₂ (Sample 26g).

FIG. 20 shows micrographs of Samples 26a-g. FIG. 20 shows that multipleimaging in the same Cy3 channel was possible by staining with differentantibodies and destroying the signal using H₂O₂.

Example 19 Residual Stain Following Staining for Abundant Proteins usingNaOH

A normal prostrate (Sample 27a) was imaged in the Cy3 channel to measurethe autofluorescence from the tissue. Sample 27a was then stained withCy3-conjugated SmA to form Sample 27b, imaged and then treated with NaOHto form Sample 27c, and imaged again. Staining, imaging, and dyedestruction steps were performed according to the procedures describedhereinabove in Examples 8 and 9. FIG. 21 shows the micrographs ofSamples 27a-c. Residual stain was observed post NaOH treatment.

Residual stain values were also monitored during the twelve channelsmultiple imaging described in Example 17. Using Gimp 2.2, average pixelintensities were collected for each background and NaOH treated imageand tabulated. FIG. 22 shows a plot of average pixel intensity of thebackground for each cycle in the imaging as well as a small image ofwhat the background looked like prior to staining. A large spike inresidual stain intensity was observed in cycle 4 as SmA, an abundantlyexpressed protein was stained in cycle 3.

Example 20 Residual Stain Following Staining for Abundant Proteins usingNaOH and H₂O₂ Treatment

Two prostate slides were stained with Cy3-directly conjugated SmA(Samples 28a and 28b). Both slides were given identical pretreatmentsteps, concentrations, antigen retrieval and the only difference wassignal-destruction method: method-one being with NaOH (Sample 28c), theother with H₂O₂ (Sample 28d). Two other prostate slides were stainedwith Cy3-directly conjugated pan cadherin (Samples 29a and 29b). Bothslides were given identical pretreatment steps, concentrations, antigenretrieval and the only difference was signal-destruction method;method-one being with NaOH (Sample 29c), the other with H₂O₂ (Sample29d).

FIG. 23 shows head to head micrographs of SmA and pan cadherin stainingand signal removal. H₂O₂ showed more efficient dye removal for both SmAand pan cadherin when compared to NaOH.

Example 21 Residual Stain Following Multiple Cycle Staining for AbundantProteins using NaOH and H₂O₂ Treatment

Two prostate slides were stained with Cy3-directly conjugated SmA(Samples 30a and 30b). Both slides were given identical pretreatmentsteps, concentrations, antigen retrieval and the only difference wassignal-destruction method; method-one being with NaOH (Sample 30c), theother with H₂O₂ (Sample 30d). The slides were subjected to 9 stainingand signal-destruction cycles before staining with Cy3-conjugated pancytokeratin.

FIG. 24 compares staining from first cycle pan cadherin stain to the9^(th) cycle of pan cytokeratin using NaOH and H₂O₂ to destroy thesignal after each staining. H₂O₂ showed more efficient dye removal after9 cycles when compared to NaOH. FIG. 25 compares background fromunstained samples (Samples 30e and 30f) and after 9 cycles of NaOH andH₂O₂ treatment (Samples 30c and 30d). H₂O₂ showed more efficient dyeremoval for both SmA and pan cadherin when compared to NaOH. FIG. 26 isa plot of average pixel intensities for the background of each cycle forthe NaOH and H₂O₂ slides. The background for the H₂O₂ slide wassignificantly less for each cycle after the initial baseline. Below eachcycle is also a small image of each background with the NaOH backgroundimage on top and H₂O₂ background image on bottom.

Example 22 Antibody Stability to Chemical Agents

A colon tissue slide was stained with a primary antibody rabbit antibeta catenin and targeted with a Cy3-conjugated donkey anti-rabbitsecondary antibody to form Sample 31a. Sample 31a was imaged and thentreated with a NaOH solution to form Sample 31b, which was imaged again.Staining, imaging, and dye destruction steps were performed according tothe procedures described hereinabove in Examples 8 and 9. Sample 31b wasrestained with a Cy3-conjugated anti rabbit secondary antibody to formSample 31c and imaged again. FIG. 27 shows micrographs of Samples 31a-c.FIG. 27 shows that the primary antibody remains bound to the sampleafter NaOH treatment.

A colon tissue slide was stained with a primary antibody mouse-anti PCNAand targeted with a Cy3-conjugated donkey anti-mouse secondary antibodyto form Sample 32a. Sample 31a was imaged and then treated with a NaOHsolution to form Sample 32b, which was imaged again. Staining, imaging,and dye destruction steps were performed according to the proceduresdescribed in Examples 8 and 9. Sample 32b was restained with aCy3-conjugated anti-mouse secondary antibody to form Sample 32c andimaged again. FIG. 28 shows that the primary antibody is still bound tothe sample after treatment with NaOH.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are thereof to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

What is claimed is:
 1. A kit for detection of multiple targets in abiological sample, comprising: multiple probes, wherein each probecomprises a binder and a fluorophore, wherein each probe is configuredto specifically bind to a target via the binder, wherein the bindercomprises a primary antibody and wherein the fluorophore is coupled to asecondary antibody that binds to the target via the primary antibody;and at least one chemical agent in a concentration that, when applied toeach probe, is configured to modify the fluorescence signal provided bythe fluorophore of at least one probe without affecting the integrity ofthe binder in the at least one probe such that the primary antibody,when bound to the target, remains bound to the target after modificationof the fluorescent signal by the at least one chemical agent underconditions in which the fluorophore is destroyed.
 2. The kit of claim 1,wherein the at least one chemical agent is configured to modify thefluorescence signal provided by the fluorophores of all of the multipleprobes.
 3. The kit of claim 1, wherein the multiple probes comprise afirst probe comprising a first binder and a first fluorophore, whereinthe first probe is configured to specifically bind to a first target,and wherein the first signal generator is configured to provide a firstfluorescence signal; and a second probe comprising a second binder and asecond fluorophore, wherein the second probe is configured tospecifically bind to a second target, and wherein the second signalgenerator is configured to provide a second fluorescence signal.
 4. Thekit of claim 3, wherein the first target is a protein and the secondtarget is a nucleic acid.
 5. The kit of claim 1, wherein the target is aprotein antigen.
 6. The kit of claim 1, further comprising amorphological stain configured to provide a distinguishable signal thatdoes not overlap with the fluorescence signal produced by thefluorophores of multiple probes.
 7. The kit of claim 6, wherein themorphological stain is a compartmental marker.
 8. The kit of claim 6,wherein the at least one chemical agent is incapable of modifying thesignal provided by the morphological stain.
 9. The kit of claim 1,wherein the at least one chemical agent is chosen from hydrogenperoxide, sodium hydroxide, or a periodate salt.
 10. The kit of claim 1,wherein the multiple probes comprises at least one probe, thefluorophore of which is configured to provide a fluorescence signal thatis not modified by the at least one chemical agent.
 11. The kit of claim1, wherein the binder is an antibody and the fluorophore is a cyaninedye.
 12. A kit for detection of multiple targets in a biological sample,comprising: a first probe, comprising a first binder and a firstfluorophore, wherein the first probe is configured to specifically bindto a first target via the first binder, and wherein the firstfluorophore is configured to provide a first fluorescence signal; asecond probe, comprising a second binder and a second fluorophore,wherein the second probe is configured to specifically bind to a secondtarget via the second binder, and wherein the second fluorophore isconfigured to provide a second fluorescence signal; a first chemicalagent in a concentration that, when applied to each probe, is configuredto modify the first fluorescence signal provided by the firstfluorophore without affecting the integrity of the first binder suchthat the first binder, when bound to the first target, remains bound tothe first target after modification of the first fluorescent signal bythe first chemical agent under conditions in which the first fluorophoreis destroyed; and a second chemical agent configured to modify thesecond fluorescence signal provided by the second fluorophore withoutaffecting the integrity of the second binder such that the secondbinder, when bound to the second target, remains bound to the secondtarget after modification of the second fluorescent signal by the secondchemical agent.
 13. The kit of claim 12, wherein the first fluorophoreis a Cy5 dye or a Cy7 dye, and the second fluorophore is a Cy3 dye. 14.The kit of claim 12, wherein the first target is a protein and thesecond target is a nucleic acid.
 15. The kit of claim 12, wherein eitherthe first chemical agent or the second chemical agent is an oxidizingagent.
 16. A kit for detection of multiple proteins in a single tissuesection, comprising: a plurality of cyanine dye-labeled antibodies; andat least one oxidizing agent in a concentration that, when applied tothe cyanine dye-labeled antibodies, is configured to modify the cyaninefluorescence of at least one of the plurality of cyanine dye-labeledantibodies without affecting the integrity of the antibodies.
 17. Thekit of claim 1, wherein the at least one chemical agent configured tomodify the fluorescence signal is capable of the modifying at roomtemperature.
 18. The kit of claim 1, wherein the at least one chemicalagent configured to modify the fluorescence signal is a buffered sodiumhydroxide solution having a pH of about
 12. 19. The kit of claim 1,wherein the at least one chemical agent configured to modify thefluorescence signal is a hydrogen peroxide solution having a volumepercentage of about 3%.
 20. The kit of claim 1, wherein the at least onechemical agent configured to modify the fluorescence signal comprises afirst solution comprising the at least one chemical agent configured tomodify the fluorescence signal and a second solution comprising the atleast one chemical agent configured to modify the fluorescence signal.21. The kit of claim 1, wherein destruction of the fluorophore does notaffect binding of the primary antibody with the secondary antibody suchthat the secondary antibody remains bound to the primary antibody afterthe destruction of the fluorophore.
 22. The kit of claim 1, wherein thechemical agent does not destroy a fluorophore associated with amorphological stain.
 23. The kit of claim 1, wherein the morphologicalstain is DAPI.
 24. The kit of claim 1, wherein the chemical agent isconfigured to destroy the fluorophore but not the morphological stainafter contact times of at least 30 minutes.
 25. The kit of claim 1,wherein the chemical agent is configured to destroy the fluorophore suchthat an average pixel intensity of an imaged sample including the targetafter the destruction is less than 7× a background average pixelintensity observed from the sample before application of thefluorophore.